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PMC1424222

9412467

JCB.29208f6

Figure 6 Colocalization of fur/f and γ-adaptin. Replicate plates of M2 ( A and C ) and A7 ( B and D ) cells were infected with the vaccinia recombinant expressing fur/f (moi = 10). At 4 h after infection, the cells were fixed with paraformaldehyde, permeabilized, and incubated with mAb M2 to detect fur/f ( A and B ) and 100/3 to detect γ-adaptin ( C and D ). The mAb M2 was visualized with anti–mouse IgG 1 -TXR and mAb 100/3 was visualized with IgG 2b -FITC.

CC BY-NC-SA

no

2022-01-13 07:10:05

J Cell Biol. 1997 Dec 29; 139(7):1719-1733

PMC1424222

9412467

JCB.29208f7

Figure 7 Time course of fur/f internalization. Parallel plates of M2 ( A , C , E , and G ) and A7 ( B , D , F , and H ) cells grown on glass coverslips were infected with VV:hfur/f (moi = 10). At 4 h after infection, mAb M1 (6 μg/ml final concentration) was added to the culture media for either 5 min ( A and B ), 15 min ( C–F ) or 60 min ( G and H ). Cells in C–F were also incubated with 40 ng/ml r-Tf and 100 nM tautomycin (to accumulate internalized furin in the early endosomes). C and D show mAb M1 and E and F show r-Tf in double-labeled cells. Samples were processed for immunofluorescence microscopy as described in Fig. 5 . Internalized mAb M1 was visualized with anti–mouse IgG 2b -TXR antibody.

CC BY-NC-SA

no

2022-01-13 07:10:05

J Cell Biol. 1997 Dec 29; 139(7):1719-1733

PMC1424222

9412467

JCB.29208f8

Figure 8 Temperature dependence of fur/f retrieval to the TGN. Replicate plates of M2 cells were infected with VV:hFur/f as above. At 4 h after infection, the cells were placed at 23°C, and then treated with mAb M1 ( A and B ) or mAb M2 ( C–F ) for 1 h and either fixed immediately ( A ) or washed to remove the mAb ( B–F ) and shifted to 37°C for an additional hour before fixation. Before fixation, the samples in E and F were treated with 5 μg BFA/ml for 20 min. After fixation, the samples in C–F were incubated with mAb 100/3 to visualize both γ-adaptin ( D and F ) and internalized fur/f (mAb M2, C and E ). Internalized mAbs M1 and 100/3 were visualized with anti–mouse IgG 2b -TXR antibody. Internalized mAb M2 was visualized with anti–mouse IgG 1 -FITC.

CC BY-NC-SA

no

2022-01-13 07:10:05

J Cell Biol. 1997 Dec 29; 139(7):1719-1733

PMC1424222

9412467

JCB.29208f9

Figure 9 Importance of ABP-280 to the localization of endocytic compartments. Parallel plates of M2 ( A , C , and E ) and A7 ( B , D, and F ) cells were grown on coverslips. Early endocytic compartments were visualized by treating the cultures with rhodamine-transferrin (r-Tf, 40 ng/ml) for 30 min at 37°C before fixation ( A and B ). Late endosomes were visualized by addition of Texas Red-dextran beads (10,000 mol wt, 10 μg/ml) for 30 min at 37°C before fixation ( C and D ). To visualize lysosomes, cells were fixed, permeabilized with detergent, and incubated with a LAMP-1 antibody followed by incubation with anti–mouse IgG 1 -FITC ( E and F ).

CC BY-NC-SA

no

2022-01-13 07:10:05

J Cell Biol. 1997 Dec 29; 139(7):1719-1733

PMC1424222

9412467

JCB.29208f10a

Figure 10 Dependence of ABP-280 on processing of furin substrates. ( A ) Processing of pro-βNGF. Parallel plates of A7 and M2 cells were infected with a vaccinia recombinant expressing mouse pro-βNGF (moi = 10). At 3 h after infection, the cells were metabolically labeled with 35 S-[Met/Cys]. Secreted βNGF proteins were immunoprecipitated from the culture media with pooled rat anti–βNGF antibodies. The washed immunoprecipitates were then resolved by SDS-PAGE and pro-βNGF-derived proteins visualized by phosphoimage analysis. ( B ) Processing of Pseudomonas exotoxin. Parallel cultures of M2 and A7 cells were incubated in culture medium containing 100 nM PE for the indicated times. The rinsed cells were then metabolically labeled with 35 S-[Met/Cys] for 30 min, harvested in mRIPA, and the incorporation of [ 35 S]-amino acids into TCA-precipitable material was quantified by scintillation counting. A7 cells, white bars; M2 cells, hatched bars.

CC BY-NC-SA

no

2022-01-13 07:10:05

J Cell Biol. 1997 Dec 29; 139(7):1719-1733

PMC1424222

9412467

JCB.29208f11

Figure 11 A model for ABP-280 on trafficking within the TGN/endosomal system. Shown are the routing of TfR, furin, the α 2 MR/ LRP-dependent internalization of PE, the proteolytic activation of the toxin in early endosomes, and the localization of late endosomes and lysosomes in the TGN/endosomal system. In ABP-280 + cells (e.g., A7), this actin binding protein directs formation of the cortical microfilaments into an orthogonal lattice, whereas, in ABP-280 − cells (e.g., M2), the actin cables form disorganized arrays. In ABP-280 + cells, lysosomes and late endocytic compartments are concentrated primarily in the paranuclear region, whereas, in ABP-280 − cells, these compartments are mislocalized (Fig. 9 ). Whether the localization of these compartments is dependent on their direct interaction with ABP-280 or microfilaments is not known. Conversely, the localization of early endosomes and the TGN is independent of ABP-280 (Figs. 5 – 9 ). TfR cycles constitutively between the early endosomes and the plasma membrane, and neither the cell surface receptor number, the cycling time of the receptor, nor the subcellular distribution of early endosomes is directly affected by the presence or absence of ABP-280 (Figs. 4 and 9 ). By contrast, the trafficking of furin is directed by ABP-280. In A7 cells, furin present at the cell surface is tethered by ABP-280 (Fig. 3 ). It is conceivable that this tethering gathers the proprotein convertase into patches to form furin processing compartments at the cell surface (e.g., activation of the anthrax toxoid [ Klimpel et al., 1992 ] and potential MT-MMP1 [see Discussion ]). Whether the α 2 MR/LRP is similarly tethered is not known. Release of furin from ABP-280 by an as yet unidentified mechanism allows the endoprotease to be retrieved into clathrin-coated pits where the tyrosine-based and/or di-leucine-like motifs within the furin cd are available to interact with the cellular internalization machinery (e.g., AP-2 complexes). In the ABP-280-deficient M2 cells, furin delivered to the cell surface is not tethered to the cortical microfilaments and instead is rapidly internalized into clathrin-coated pits. This unrestricted internalization of furin enhances the probability that it encounters substrates (e.g., PE) in endocytic compartments. Although ABP-280 modulates the rate of furin internalization (Fig. 4 ), it does not effect the efficiency of sorting of the endoprotease from the cell surface to early endosomes (Fig. 7 ). By contrast, the efficient retrieval of the internalized furin from the early endosomes to the TGN requires ABP-280 (Figs. 5 and 7 ). Large arrows represent efficient sorting, whereas small arrows denote inefficient sorting.

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no

2022-01-13 07:10:05

J Cell Biol. 1997 Dec 29; 139(7):1719-1733

PMC1435730

14691139

200306033f1

Figure 1. The spine morphogenesis in cultured hippocampal neurons is failed in absence of the EphB receptors. (A) Expression of EphB1, EphB2, and EphB3 in cultured hippocampal neurons from the various combinations of EphB KOs by Western blot analysis. (B) Morphology of GFP-labeled spines in 21 DIV–cultured hippocampal neurons from WT and triple EphB-deficient (EphB1−/−,EphB2−/−, EphB3−/−) mice. The hippocampal neurons were transfected with GFP at 7 DIV and examined at 21 DIV. Bars: (top) 10 μm; (bottom) 2 μm. (C) Quantitative analysis of the lengths of dendritic protrusions ( n > 500).

CC BY-NC-SA

no

2022-01-13 07:22:20

J Cell Biol. 2003 Dec 22; 163(6):1313-1326

PMC1435730

14691139

200306033f2

Figure 2. Triple EphB-deficient neurons fail to make spine synapses in cultures. Cultured hippocampal neurons from WT and triple EphB-deficient (EphB1−/−,EphB2−/−,EphB3−/−) mice were transfected with GFP at 7 DIV and examined at 21 DIV. (A) Detection of polymerized F-actin by rhodamine-coupled phalloidin. Confocal images of GFP fluorescence (green) and rhodamine-coupled phalloidin (red). (B) Immunodetection of synaptophysin-positive presynaptic boutons. Confocal images of GFP fluorescence (green) and anti-synaptophysin IR (red). (C) Analysis of the distribution of post-synaptic sites by immunodetection of post-synaptic protein PSD-95. Confocal images of GFP fluorescence (green) and anti–PSD-95 IR (red). (D) Detection of glutamatergic synapses by immunostaining with anti-GluR2 and NMDAR2A/B antibodies. Confocal images of GFP fluorescence (green) and anti-GluR2 (AMPAR) or anti-NMDAR2A/B (NMDAR) IRs (red). (E) Detection of GABAergic synapses by immunostaining for GAD. Confocal images of GFP fluorescence (green) and anti-GAD65 IR (red). (F) Western blot analysis of subcellular distribution of NMDAR2A/B, GluR2, PSD-95 and synaptophysin in WT (left) and triple EphB1−/−EphB2−/−EphB3−/− (right) hippocampal neurons at 21 DIV. Subcellular fractionations were prepared as described in Materials and methods. Bars, 1 μm.

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no

2022-01-13 07:22:20

J Cell Biol. 2003 Dec 22; 163(6):1313-1326

PMC1435730

14691139

200306033f3

Figure 3. The EphB1 and EphB2 receptors are main contributors to spine morphogenesis in the cultured hippocampal neurons. Analysis of spine morphogenesis in cultured hippocampal neurons from WT and EphB-deficient mice: single EphB KOs (EphB1−/−, EphB2−/−, or EphB3−/−); double EphB KOs (EphB1−/−;EphB2−/−, or EphB2−/−;EphB3−/−); triple EphB KO (EphB1−/−;EphB2−/−;EphB3−/−), or double mutants that express a truncated form of the EphB2 (EphB1−/−;EphB2lacZ). The cultured hippocampal neurons were transfected with GFP at 7 DIV. Spine morphology was examined at 21 DIV. (A) Confocal images of GFP-labeled spines in 21 DIV–cultured hippocampal neurons with various EphB KOs. Bars, 1 μm. (B) Quantifications of spine morphogenesis and synapses in 21 DIV–cultured hippocampal neurons with various EphB KOs. The spine morphogenesis was assayed by analysis of spine lengths (left; n > 500), percent of mature mushroom-like spines relative to total number of protrusions (right, top). Synapse formation was analyzed by numbers of synaptophysin-positive and PSD-95–positive IRs (right, bottom). Error bars indicate SD. *, P < 0.05; **, P < 0.001; ***, P < 0.0001 with t test.

CC BY-NC-SA

no

2022-01-13 07:22:20

J Cell Biol. 2003 Dec 22; 163(6):1313-1326

PMC1435730

14691139

200306033f4

Figure 4. Distribution of ephrin B and EphB receptors in cultured hippocampal neurons. (A) Immunodetection of EphB1, EphB2, and EphB3 in 21 DIV–cultured hippocampal neurons. Confocal images show partial colocalization of the EphB1 and EphB2 or EphB2 and EphB3 IRs (yellow). Bars: (top) 10 μm; (bottom) 1 μm. (B) Double immunolabeling of 21 DIV–cultured hippocampal neurons with anti–pan-ephrin-B (green) and anti-synaptophysin (left, red) or anti-NF antibody (right, red). The confocal analysis show axon-specific localization of the B-class ephrins. Bars, 10 μm. The examples of juxtaposition of synaptophysin and ephrin-B are indicated by arrows. (C) B-class ephrins (red) and EphB2 (green) IRs are localized in close proximity, but do not overlap (no yellow, arrows). Bars: (left) 10 μm; (right) 1 μm.

CC BY-NC-SA

no

2022-01-13 07:22:20

J Cell Biol. 2003 Dec 22; 163(6):1313-1326

PMC1435730

14691139

200306033f5

Figure 5. Clustered ephrin-B2-Fc promotes spine morphogenesis in cultured hippocampal neurons. Clustered ephrin-B2-Fc or control Fc was applied to cultured GFP-expressing hippocampal neurons at (A–C) 7 DIV or (D–H) 14 DIV. (A) Immunodetection of EphB2 in 7 DIV–cultured hippocampal neurons after treatment with control Fc (left) or EphrinB2-Fc (right). Clustered ephrin-B2-Fc induced clustering of the EphB2. Bars, 10 μm. (B) Autophosphorylation of EphB2 is induced by ephrin-B2-Fc treatment as shown by Western blot. (C) Live images of GFP-labeled dendritic protrusions in 7 DIV WT hippocampal neurons after treatment with ephrin-B2-Fc, or Fc at 0 and 25 min. Bars, 1 μm. (D) Confocal images of spines in GFP-labeled 14 DIV WT hippocampal neurons treated with ephrin-B2-Fc (left) or control Fc (right) for 4 h. The spines are visualized by GFP fluorescence (green) and actin polymerization (red) by staining for F-actin with rhodamine-coupled phalloidin. Filopodia are indicated by arrows; spines with smaller heads are indicated by open arrowhead; and spines with large heads are indicated by closed arrowheads. Bars, 1 μm. (E) Quantitative analysis of the lengths of dendritic protrusions in 14 DIV–cultured hippocampal neurons, WT (top) or triple EphB-deficient (EphB1B2B3−/−, bottom), treated with ephrin-B2-Fc (left) or control Fc (right). Group A represents spines and group B represents dendritic filopodia ( n > 500). Note: Clustered ephrin-B2-Fc induced elimination of dendritic filopodia and increased spine number in 14 DIV–cultured hippocampal neurons from WT mice, but not in triple EphB-deficient hippocampal neurons. (F–H) Confocal images of spines in GFP-labeled 14 DIV WT hippocampal neurons treated with ephrin-B2-Fc (F) or control Fc (G), and triple EphB1,B2,B3 KOs hippocampal neurons treated with ephrin-B2-Fc (H). Bars: (top) 10 μm; (bottom) 1 μm. (F) Clustered ephrin-B2-Fc induced mature mushroom-like morphology and EphB2 clustering in WT 14 DIV–cultured hippocampal neurons. The cultures are analyzed for spine morphology by GFP fluorescence (green) and for EphB2 clusters by immunostaining using anti-EphB2 antibody (red). Note: The spines with bigger heads (arrows), which were induced by ephrin-B2-Fc, showed EphB2 clusters (red, high magnification insert). (G) Control Fc treated cultures show only few EphB2 clusters (red, arrows) that are typical for hippocampal neurons at 14 DIV. (F and G) Filopodia are indicated by arrowheads; spines are indicated by arrows. (H) Clustered ephrin-B2-Fc does not induce mushroom-like morphology in triple EphB-deficient 14 DIV hippocampal neurons (EphB1B2B2−/−). The EphB2 IR is not detected.

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no

2022-01-13 07:22:20

J Cell Biol. 2003 Dec 22; 163(6):1313-1326

PMC1435730

14691139

200306033f6ac

Figure 6. Spines in hippocampi of triple EphB-deficient mice reveal abnormal headless or small-headed morphology. (A) A projection of a 3-D reconstructed confocal image of biocytin-filled CA3 pyramidal cell pair from hippocampus of the triple EphB-deficient (EphB1B2B3−/−) mice (left). (Right) High magnification view shows dendrites and spines. Bars: (left) 100 μm; (right) 10 μm. (B) Morphology of spines in biocytin-filled CA3 neurons of hippocampal slices from triple EphB-deficient (EphB1B2B3−/−) and WT mice. There are fewer spines are found in triple EphB-deficient neurons as compared with WT. The spines in EphB-deficient neurons exhibit headless (arrow) or small-headed morphology (arrowhead), whereas in WT neurons spines reveal mature mushroom-like morphology with bulbous heads and thin necks (right). Bars, 2 μm. (C) Quantification of spine head area (left) and spine lengths (right) in triple EphB-deficient (EphB1B2B3−/−) and WT hippocampus ( n > 300 for each mice). Error bars indicate SD. *, P < 0.05; **, P < 0.001; ***, P < 0.0001 with t test. (D and E) Immunohistochemical localization of (D) PSD-95 and (E) synaptophysin in stratum lucidum (SL) and stratum radiatum (SR) of hippocampi in the triple EphB-deficient (EphB1/B2/B3) and WT mice (WT). SP, stratum pyramidale. Bars, 50 μm.

CC BY-NC-SA

no

2022-01-13 07:22:20

J Cell Biol. 2003 Dec 22; 163(6):1313-1326

PMC1435730

14691139

200306033f7

Figure 7. Multiple EphB receptors are responsible for spine morphogenesis in hippocampus in vivo. (A) Spine morphology in biocytin-filled CA1 and CA3 neurons of hippocampal slices from WT and EphB-deficient mice: double EphB KOs (EphB1−/−;EphB2−/−, or EphB2−/−;EphB3−/−); triple EphB KO (EphB1−/−;EphB2−/−;EphB3−/−), or mutant that express a truncated form of the EphB2 (EphB2−/−;EphB2lacZ). Bars, 2 μm. (B) Quantifications of spine density (top, left), spine lengths (top, right), percent of mature spines relative to total number of protrusions (bottom, left), and spine head area (bottom, right). Mature spines are defined as mushroom-shaped spines with large heads or stubby spines. Error bars indicate SD. *, P < 0.05; **, P < 0.001; ***, P < 0.0001 with t test.

CC BY-NC-SA

no

2022-01-13 07:22:20

J Cell Biol. 2003 Dec 22; 163(6):1313-1326

PMC1435730

14691139

200306033f8

Figure 8. The post-synaptic component and PSD area of asymmetric synapses (spines) are significantly reduced in the triple EphB-deficient hippocampus. Representative electron micrographs of ultrathin sections of CA3 hippocampus from triple EphB-deficient (EphB1B2B3−/−) or WT mice. (A) Representative electron micrograph of longitudinal section through the dendrite shows increased number of symmetric synapses on dendritic shaft (arrows) in triple EphB-deficient neurons (EphB1B2B3−/−), as compared with WT. Note: The dendrite is ensheathed by astrocytic processes (blue). Bar, 1 μm. (B) Representative electron micrographs that show cross-sectional areas of post-synaptic component (asterisks) and PSD of asymmetric synapses in CA3 hippocampus of triple EphB-deficient (EphB1B2B3−/−) or WT mice. Bars, 1 μm. (C) Quantification of cross-sectional area of post-synaptic component (left) and PSD area (right) of asymmetric synapses (spines) in hippocampi of triple EphB-deficient (EphB1B2B3−/−) and WT mice. (D) Quantification of cross-sectional area of post-synaptic component (left, post-synaptic area) and PSD length of asymmetric synapses (right) in CA1 and CA3 areas of hippocampi from WT and the EphB-deficient mice: double EphB KOs (EphB1−/−; EphB2−/−, or EphB2−/−;EphB3−/−); and triple EphB KO (EphB1−/−;EphB2−/−; EphB3−/−). (C and D) Error bars indicate SD. *, P < 0.05; **, P < 0.001; ***, P < 0.0001 with t test.

CC BY-NC-SA

no

2022-01-13 07:22:20

J Cell Biol. 2003 Dec 22; 163(6):1313-1326

PMC1435730

14691139

200306033f9

Figure 9. Schematic representation of spines in triple EphB-deficient hippocampal neurons in vitro and in vivo. In vitro , WT hippocampal neurons form spines with asymmetric synapses and large heads containing polymerized actin and large PSD. The dendrites are not ensheathed by astrocytic processes. Small number of filopodia is found in mature neurons. In vitro , triple EphB1B2B3 − / − hippocampal neurons don't form spines. There are many filopodia are formed and only symmetric synapses on dendritic shaft are found. The dendrites are not ensheathed by astrocytic processes. In vivo , WT hippocampal neurons form spines with asymmetric synapses, bulbous heads, and large PSD. The dendrites and synapses are enwrapped by astrocytic processes. Filopodia are usually not found in mature neurons. In vivo, triple EphB1B2B3 − / − hippocampal neurons form fewer spines. These spines have immature small-headed or headless morphology and reduced PSD length. The dendrites and synapses are enwrapped by astrocytic processes. Small number of filopodia is found in mature neurons.

CC BY-NC-SA

no

2022-01-13 07:22:20

J Cell Biol. 2003 Dec 22; 163(6):1313-1326

PMC1459911

16275765

20050953f1

Figure 1. Liver-derived NKT cells are optimal mediators of MCA-1 tumor immunosurveillance. TCR Jα18 −/− mice were inoculated s.c. in the right hind flank with 10 5 MCA-1 cells. Groups of TCR Jα18 −/− mice then received sorted liver- (A), spleen- (B), or thymus-derived (C) NKT cells or, 2% NMS in PBS by way of i.v. injection. Sorted populations were always ≥94%, ≥91%, and ≥97% pure, respectively. Results were recorded every other day as the mean tumor size (cm 2 ) ± SEM. Data are pooled from three (A), one (B), and two (C) experiment(s) with three to five mice/group/experiment. The number of mice in each group is indicated in parentheses. PBS-treated TCR Jα18 −/− control data are the same for A, B, and C and is shown for comparison. Significant difference in tumor growth rate was determined between the PBS-treated TCR Jα18 −/− control group and the mice that received liver, spleen, or thymus-derived NKT cells using a Kruskal-Wallis statistical test, followed by a Dunn's post test. ***P ≤ 0.001.

CC BY-NC-SA

no

2022-01-13 09:01:09

J Exp Med. 2005 Nov 7; 202(9):1279-1288

PMC1459911

16275765

20050953f2

Figure 2. Liver-derived CD4 − NKT cells are the most potent mediators of MCA-1 tumor immunosurveillance. TCR Jα18 −/− mice were inoculated s.c. in the right hind flank with 10 5 MCA-1 cells. Groups of TCR Jα18 −/− mice then received sorted CD4 + or CD4 − liver-derived NKT cells, or 2% NMS in PBS (A), or a range of liver-derived CD4 − NKT cell doses (2.5 × 10 5 , 10 5 or 5 × 10 4 cells), by way of i.v. injection (B). Results were recorded every other day as the mean tumor size (cm 2 ) ± SEM. Data are pooled from two independent experiments (A and B). We used three to five mice/group/experiment. The number of mice with measurable tumors over total mice per group is indicated in parentheses. PBS-treated TCR Jα18 −/− control data are the same for A and B, and are shown for comparison. Significant difference in tumor growth rate was determined between the PBS-treated control group and mice receiving CD4 + NKT cells, using a Mann-Whitney U test. *P < 0.05.

CC BY-NC-SA

no

2022-01-13 09:01:09

J Exp Med. 2005 Nov 7; 202(9):1279-1288

PMC1459911

16275765

20050953f3

Figure 3. Thymus-derived NKT cells detected several days after transfer. NKT cell–enriched thymocytes were CFSE-labeled and transferred into WT recipients by way of i.v. injection (A, left). 6 d after transfer, organs were harvested and screened for the presence of CFSE + CD1d/α-GalCer + cells (A, right). These cells were gated on, and analyzed for, CD4 expression (B, middle and right). Some mice received CFSE-labeled cells that were labeled with anti-CD4 before transfer (B, right). Data are representative of six (A) or two (B) experiments with one or two mice/group.

CC BY-NC-SA

no

2022-01-13 09:01:09

J Exp Med. 2005 Nov 7; 202(9):1279-1288

PMC1459911

16275765

20050953f4

Figure 4. Liver-derived NKT cells are most efficient mediators of α -GalCer–induced antitumor immunity. TCR Jα18 −/− mice were inoculated with 5 × 10 5 B16F10 cells by way of i.v. injection. 3 h later, some mice received unfractionated liver-derived lymphocytes (A and B), NKT cell–enriched thymocytes or splenocytes, sorted thymus-derived NKT cells (A), or sorted liver- or thymus-derived CD4 + or CD4 − NKT cell subsets (B) by way of i.v. injection. On the same day as lymphocyte transfer, and again on days 4 and 8, some mice also received 2 μg α-GalCer or vehicle by way of i.p. injection. 14 d after tumor inoculation, lungs were harvested, and B16F10 colonies were counted and recorded as the mean number ± SEM. Data are pooled from two independent experiments and the total number of mice in each group is indicated in parentheses. Significant difference in tumor growth rate was determined between mice receiving NKT cells and vehicle, and mice receiving NKT cells and α-GalCer, using a Kruskal-Wallis test, followed by a Dunn's post test. *P < 0.05; **P < 0.01; ***P ≤ 0.001.

CC BY-NC-SA

no

2022-01-13 09:01:09

J Exp Med. 2005 Nov 7; 202(9):1279-1288

PMC1459911

16275765

20050953f5

Figure 5. Cytokine production by NKT cell subsets after α -GalCer stimulation. WT mice received 2 μg α-GalCer or vehicle by way of i.p. injection and were killed 2 or 48 h later (A). Liver- and spleen-derived lymphocytes were cultured without further stimulation for 2 h in Brefeldin A. After culture, cells were labeled with surface mAb, fixed, and permeabilized for intracellular cytokine staining. Cells were analyzed by flow cytometry, and CD1d/ α-GalCer + αβTCR + cells were gated on and CD4 + and CD4 − subsets screened for intracellular IFN-γ production. Gates were set based on the isotype control at each time point. (B) Liver lymphocytes and NKT cell–enriched thymocytes (with sorted spleen-derived DCs) were cultured for 12 h with 100 ng/ml α-GalCer. Brefeldin A was added for the final 2 h of culture. After culture, cells were prepared for intracellular cytokine staining (as for A). NK1.1 + αβTCR + cells from the liver and NK1.1 + cells from the thymus were electronically gated and CD4 + and CD4 − subsets were analyzed for intracellular IFN-γ production (filled line graph: DCs with α-GalCer; dotted line: nonstimulated control).

CC BY-NC-SA

no

2022-01-13 09:01:09

J Exp Med. 2005 Nov 7; 202(9):1279-1288

PMC1459911

16275765

20050953f6b

Figure 6. NKT cell–derived IL-4 production impairs antitumor function of thymus-derived NKT cells. (A) TCR Jα18 −/− mice were inoculated with 5 × 10 5 B16F10 cells by way of i.v. injection. 3 h later, some mice received unfractionated liver-derived lymphocytes, NKT cell–enriched thymocytes, or sorted liver- and thymus-derived NKT cells from WT and IL-4 −/− donors by way of i.v. injection. For IL-10 neutralization, some mice received 400 μg anti–IL-10 on the same day as lymphocyte transfer (day 0), and again on days 4 and 8 by way of i.p. injection. Some mice also received 2 μg α-GalCer or vehicle by way of i.p. injection on days 0, 4, and 8. 14 d after tumor inoculation, lungs were harvested, and B16F10 colonies were counted and recorded as the mean number ± SEM. The number of mice in each group is indicated in parentheses. Results from mice receiving WT or IL-4 −/− NKT cells from thymus or liver were compared using a Mann-Whitney rank sum test. *P < 0.05. (B) WT and IL-4 −/− mice received 2 μg α-GalCer or vehicle by way of i.p. injection and were killed 2 or 48 h later. Liver-derived lymphocytes were cultured without further stimulation for 2 h in Brefeldin A, before being labeled with surface mAb, fixed, and permeabilized for intracellular cytokine staining. Cells were analyzed by flow cytometry and CD1d/α-GalCer + αβTCR + cells were electronically gated and analyzed for intracellular IFN-γ production and CD4 expression.

CC BY-NC-SA

no

2022-01-13 09:01:09

J Exp Med. 2005 Nov 7; 202(9):1279-1288

PMC1464409

16717281

gkl375f1

Figure 1 Structures of the chimeric proteins between c-Fos and HBZ and their effect on the trans -activation by c-Jun. Schematic representations of c-Fos, HBZ, and the various chimeras are shown; the chimeric proteins possess one or several activation domains (AD) and a bZIP structure including the DNA-binding domain (from residue 140 to 163) and the zipper (from 164 to 200). Expression plasmids of c-Fos, HBZ, and their chimeras were cotransfected with c-Jun expression plasmid together with a vector containing the luciferase gene driven by the collagenase promoter as described in the legend of Figure 2 . The results are indicated on the right. Symbols: +++++, activation above 800-fold; +++, about 200-fold; +, about 40-fold; -, inhibition of c-Jun activity.

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no

2022-01-12 17:40:49

Nucleic Acids Res. 2006 May 22; 34(9):2761-2772

PMC1464409

16717281

gkl375f2

Figure 2 Characterization of the amino acid region involved in the differential activation of c-Jun by c-Fos and HBZ. ( A ) Expression of the chimeras HBZ122/Fos and HBZ209/Fos in vivo . Expression of the chimeric proteins in 293T cells was detected by western blotting using the mouse anti-Myc antibody (NT: not transfected). Molecular size markers (kDa) are shown on the left and migration of the chimeras is indicated by the arrow on the right. ( B and C ) Analysis of the trans -activation by the chimeras in the presence of c-Jun. CEM cells were cotransfected with 2 μg of a vector containing the luciferase reporter gene driven by the collagenase promoter, 5 μg of pcDNA3.1- lacZ (β-galactosidase-containing reference plasmid), 1 μg of pcDNA-c-Jun and 2 μg of the vector pcDNA3.1(−)/Myc-His expressing each of the tested chimera. Luciferase values are expressed as folf increases relative to values measured in cells transfected with empty pcDNA3.1(−)/Myc-His in the presence of the luciferase reporter vector. The total amount of DNA in each series of transfection was equal, the balance being made up with the empty plasmids. Luciferase values were normalized for β-galactosidase activity. Values represent the mean ± SD ( n = 3).

CC BY-NC

no

2022-01-12 17:40:49

Nucleic Acids Res. 2006 May 22; 34(9):2761-2772

PMC1464409

16717281

gkl375f3

Figure 3 Comparison of the amino acid region adjacent to the bZIP domain between HBZ and c-Fos. The HBZ and c-Fos DNA-binding domains were aligned by using the basic motif and leucine zipper as a reference point. Numbering is relative to the +1 methionine of the chimeras shown in Figure 1 . ( A ) The cluster of six charged residues studied in the paper (134–139 amino acids) is in bold and the leucine residues of the zipper are in boxes. ( B ) Comparison of the amino acid sequences adjacent to the leucine zipper of HBZ, c-Fos, and the different mutants produced from HBZ163/ZIPFos. The mutated residues are bold and the basic motifs of the DNA-binding domains (142–157 amino acids) are in a box. The conserved alanine and serine/cysteine residues of the basic motif correspond to position 149 and 153.

CC BY-NC

no

2022-01-12 17:40:49

Nucleic Acids Res. 2006 May 22; 34(9):2761-2772

PMC1464409

16717281

gkl375f4

Figure 4 The EEEEKR sequence immediately adjacent to the bZIP domain of c-Fos confers a trans -activation potential to heterodimerized c-Jun. ( A ) The transcriptional activity of the mutated chimeras were analyzed as described in the legend of Figure 2 . Bars labeled as EQERRE, EEEERE, EEEERR and EEEEKR correspond to cells tranfected by HBZ140/bZIPFos, the two mutated chimeras produced from HBZ140/bZIPFos, and HBZ130/bZIPFos, respectively. For comparison, luciferase activity was also measured from cells transfected with pcDNA-c-Jun alone or in the presence of the wild-type HBZ expression vector. ( B ) Expression of the chimeric proteins in 293T cells was detected by western blotting using the mouse anti-Myc antibody as already mentioned. Migration of the chimeras is shown by the arrow and the asterisks indicate non-specific bands. ( C ) Interaction study between the generated chimeras with c-Jun by yeast two-hybrid assay using a liquid culture β-galactosidase assay. Yeasts were transformed with the expression vector pGAD containing the entire coding sequence of c-Jun fused to the GAL4 activation domain along with pGBT9 expressing the GAL4 DNA-binding domain fused to the region encompassing residues 123 to 209 from c-Fos, HBZ, or the chimeras. The β-galactosidase was carried out on three independent colonies per transformation assay using ONPG as substrate. Mean values presented in the graph are expressed in Miller units. ( D ) DNA-binding activity of chimeras. Microwells containing the AP-1 binding probe were incubated with nuclear cell extracts of 293T cells cotransfected with 4 μg of pcDNA-c-Jun and 4 μg of the vector expressing c-Fos, HBZ, HBZ140/bZIPFos (EQERRE) or its mutated form (EEEERR). The negative and positive controls correspond to cells transfected by the pcDNA empty vector and pcDNA-c-Jun alone. The data represent the means of three values ± S.D. Above, immunoblotting of nuclear proteins from transfected 293T cells using anti-c-Jun antibodies.

CC BY-NC

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2022-01-12 17:40:49

Nucleic Acids Res. 2006 May 22; 34(9):2761-2772

PMC1464409

16717281

gkl375f5

Figure 5 Activity analysis of c-Fos substitution mutants in the HBZ DNA-binding domain. ( A ) The transcriptional activity and ( B ) in vivo expression were analyzed as described in the legend of Figure 2 . ( C ) The interaction with c-Jun and ( D ) the DNA-binding activity of H3F and H14F mutants were tested as described in the legend of Figure 4 .

CC BY-NC

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2022-01-12 17:40:49

Nucleic Acids Res. 2006 May 22; 34(9):2761-2772

PMC1464409

16717281

gkl375f6

Figure 6 Subcellular localization of HBZ, c-Fos and the different chimeras in COS cells. Expression vectors for HBZ, c-Fos and the different chimeras were transiently transfected into COS cells. Cells were cultivated on glass sides, fixed, and stained with the Hoechst solution. The localization of the Myc-tagged chimeras was analyzed by immunofluorescence microscopy using the mouse anti-Myc antibody and goat anti-mouse IgG antibodies coupled to FITC. The blue fluorescence of the nuclei results from ultraviolet (UV) illumination of the fixed cells.

CC BY-NC

no

2022-01-12 17:40:49

Nucleic Acids Res. 2006 May 22; 34(9):2761-2772

PMC1464409

16717281

gkl375f7

Figure 7 The EQERRE sequence immediately adjacent to the bZIP domain of HBZ confers a trans -activation potential to JunD. In vivo expression of the HBZ-mutMD protein ( A ) and its effect on c-Jun ( B ) or JunD ( C ) DNA binding (on the left) and transcriptional (on the right) activities were analyzed as described in the legends of the other figures.

CC BY-NC

no

2022-01-12 17:40:49

Nucleic Acids Res. 2006 May 22; 34(9):2761-2772

PMC1464409

16717281

gkl375f8

Figure 8 The modulatory domain of c-Fos affects Jun transcriptional potency. The transcriptional activity of c-Fos-mutMD was analyzed in the presence of c-Jun, JunB or JunD. For comparison, luciferase activity was also measured from cells transfected with the wild-type c-Fos expression vector. CEM cells were cotransfected with 2 μg of a vector containing the luciferase reporter gene driven by the collagenase promoter, 5 μg of pcDNA3.1- lacZ , 1 μg of Jun expression vector alone or with 1 μg of pcDNA-c-Fos or pcDNA-c-Fos-MutMD. Luciferase values are expressed as fold increases relative to values measured in cells transfected with the luciferase reporter vector alone. The total amount of DNA in each series of transfection was equal, the balance being made up with the empty plasmids. Luciferase values were normalized for β-galactosidase activity. Values represent the mean ± SD ( n = 3).

CC BY-NC

no

2022-01-12 17:40:49

Nucleic Acids Res. 2006 May 22; 34(9):2761-2772

PMC1472625

0

gkl399f1

CC BY-NC

no

2022-01-12 14:27:15

Nucleic Acids Res. 2006 May 24; 34(9):2845

PMC1474057

16738136

gkl371f1

Figure 1 Selected clusters of ncRNA expression profiles. ncRNAs within functional groups usually showed similar expression patterns. The cluster numbers are identical to Supplementary Figure 4. An asterix indicates that the ncRNA has more than one locus in the genome. ( a and b ) Two clusters dominated by Sm Y/snlRNAs. ( c and d ) Clusters dominated by SL2 RNAs. ( e ) A group of snoRNAs that showed high expression levels at early developmental stages.

CC BY-NC

no

2022-01-12 14:27:35

Nucleic Acids Res. 2006 May 31; 34(10):2976-2983

PMC1474057

16738136

gkl371f2

Figure 2 Predicted methylation and pseudouridylation guide duplex between snoRNAs and snRNAs. The snoRNA sequences in a 5′–3′ orientation are shown in the upper strands, while snRNA sequences in a 3′–5′ orientation are shown in the lower strands. Sequence motifs are boxed and the positions of modification sites are indicated by arrows and numbers. The upper parts of the hairpins of the H/ACA snoRNAs are represented by continuous lines. ( a ) Predicted methylation guide duplex between C/D box snoRNA CeN128 and U5 snRNA. ( b and c ) Predicted pseudouridylation guide duplex between H/ACA box snoRNAs CeN104 and CeN94 and U6 snRNA.

CC BY-NC

no

2022-01-12 14:27:35

Nucleic Acids Res. 2006 May 31; 34(10):2976-2983

PMC1474057

16738136

gkl371f3

Figure 3 Relationship between intronic ncRNA and host gene expression levels. The values were normalized signal intensities for Cy-labelled samples from mixed C.elegans populations. ( a ) Expression levels of intronic ncRNA genes with either no upstream motif (Non-UM) or with upstream motif 1 (UM1) or 2 (UM2). ncRNAs with UM1 and UM2 showed no obvious correlation with their host gene mRNAs, while the non-motif ncRNAs showed a positive correlation with their host gene mRNAs. ( b ) Host gene function influenced the expressional relationship between ncRNA and host mRNA. The correlation between the expression levels of ribosomal host genes (Ribo) and their intronic ncRNAs was higher ( r = 0.83) than that of non-ribosomal hosts (Non-ribo) and their intronic ncRNAs ( r = 0.39).

CC BY-NC

no

2022-01-12 14:27:35

Nucleic Acids Res. 2006 May 31; 34(10):2976-2983

PMC1474058

16738138

gkl380f1

Figure 1 Impact of the polλDN form expression on intrachromosomal NHEJ. ( A ) Substrate used to measure NHEJ. The only expressed gene is H2-Kd, which is under control of the pCMV promoter. CD8 is not expressed because it is in inverted orientation. CD4 is not expressed because it is too far from the pCMV promoter. Two I-Sce-I sites are present in non-coding sequences. After cleavage by I-Sce-I, the internal H2-Kd/CD8 fragment is excised. Re-joining of the DNA ends can lead to two different measurable events, deletion which leads to expression of the CD4 gene and inversion which leads to expression of the CD8 gene. ( B and C ) Representation of the sequences of the I-Sce-I restriction sites in the C′10 and A′7H cell lines, respectively. In the C′10 cells the two I-Sce-I sites are in direct orientation whereas in the A′7H cells the two I-Sce-I sites are in inverted orientation (upper panel). Evaluation of the frequency of the deletion (CD4) and inversion (CD8) events in the different cell lines (median panel). Results are the mean +/− SD of three independent experiments. Asterisks represent significant statistical difference ( P < 0.05). Western blot for polλ expression in the different cell lines (lower panel).

CC BY-NC

no

2022-01-12 14:27:35

Nucleic Acids Res. 2006 May 31; 34(10):2998-3007

PMC1474058

16738138

gkl380f2

Figure 2 Effect of the expression of the polβ and polμ inactive form on NHEJ events. ( A and B ) Representation of the sequences of the I-Sce-I restriction sites in the C′10 and A′7H cell lines, respectively. In the C′10 cells the two I-Sce-I sites are in direct orientation whereas in the A′7H cells the two I-Sce-I sites are in inverted orientation (upper panel). Evaluation of the frequency of the deletion (CD4) and inversion (CD8) events in the different cell lines (median panel). Results are the mean +/− SD of three independent experiments. Western blots for polβ and polμ expression in the different cell lines (lower panel).

CC BY-NC

no

2022-01-12 14:27:35

Nucleic Acids Res. 2006 May 31; 34(10):2998-3007

PMC1474058

16738138

gkl380f3

Figure 3 Sequencing of the repair junctions. ( A ) Examples of sequences of the repair junctions of the A′7H cell line expressing the different forms of polλ. The structures of the implicated DNA ends are shown at the top of the figure. Black squares, location of the microhomologies. ( B ) Models for the different classes of end-joining events involving the four protruding nucleotides of the I-Sce-I cleavage site, using microhomology. ( C ) Percentages of the different types of NHEJ junction in the polλ WT and polλDN expressing cells.

CC BY-NC

no

2022-01-12 14:27:35

Nucleic Acids Res. 2006 May 31; 34(10):2998-3007

PMC1474058

16738138

gkl380f4

Figure 4 Cellular sensitivity to DNA damaging agents of the polλDN expressing cells. ( A ) Western blot analysis of the expression of the different forms of polλ in the DRA10 cell line. DRA10 is the parental cell line, CT2B is a cell line expressing the empty vector, R2 and R15 are independent cell lines overexpressing the WT form of polλ, RD10 and RD14 are independent cell lines expressing the polλDN form. ( B ) Cell survival after ionizing radiation and ( C ) Comparison of cell survival after ionizing radiation of polλDN-transfected cells with the NHEJ-defective cells, XR-1 ( xrcc4 mutant cell line) and the XRCC4-complemented cell line (X4V). ( D ) Homologous recombination activity. ( E ) Cell survival after treatment with mitomycin C. ( F ) Cell survival after camptothecin treatment. ( G ) Topoisomerase I expression in the different polλ expressing cell lines. All cell survival and homologous recombination results are the mean +/− SEM of 3 independent experiments.

CC BY-NC

no

2022-01-12 14:27:35

Nucleic Acids Res. 2006 May 31; 34(10):2998-3007

PMC1474058

16738138

gkl380f5

Figure 5 Chromosomal aberrations induced by IR in polλDN expressing cells. Photographs of metaphase spreads of polλDN expressing cells 24 h after treatment with IR at 2 Gy. (b, break; t, triradial).

CC BY-NC

no

2022-01-12 14:27:35

Nucleic Acids Res. 2006 May 31; 34(10):2998-3007

PMC1474060

16738141

gkl378f1

Figure 1 Pes1 mutagenesis and conditional Pes1 expression. ( A ) A panel of Pes1 deletion mutants (M1–M8) were constructed. Recombinant Pes1 wt and mutant proteins carry a C-terminal HA-tag. NLS: classical nuclear localization sequence; the N-terminal NLS consists of three overlapping NLS; bip. NLS: six overlapping bipartite NLS; BRCT: BRCT domain; acidic domains: two glutamic acid rich regions; ΨKXE: consensus SUMOylation site; HA: hemagglutinin-tag. Mutant M8 has replaced Lys517 by Arg. ( B ) Pes1 and its mutants were expressed in rat fibroblasts (TGR-1) stably transfected with the inducible EBV-based vector pRTS-1. pRTS features a bidirectional promoter expressing simultaneously the gene of interest and EGFP. After induction by doxycycline, the vector switches from active silencing to transactivation. ( C ) EGFP expression was induced in >95% of cells 24 h after addition of doxycycline, as determined by FACS analysis. ( D ) Western blot analysis of wt or mutant Pes1 proteins in TGR-1 fibroblasts by anti-HA antibodies (3F10). The mock cell line expresses luciferase instead of Pes1-HA. The indicated cell lines were treated with 1 μg/ml doxycycline for 30 h (+) or left untreated (−). Bottom panel shows Ponceau S staining as a loading control.

CC BY-NC

no

2022-01-12 14:27:35

Nucleic Acids Res. 2006 May 31; 34(10):3030-3043

PMC1474060

16738141

gkl378f2

Figure 2 Cellular localization of wt and mutant Pes1 proteins by indirect immunofluorescence. TGR-1 cells transfected with the indicated constructs were fixed with methanol/acetone after induction of the Pes1-HA alleles with doxycycline for 30 h. The HA-tagged proteins were stained by anti-HA antibodies (3F10), the nuclei were counterstained with DAPI.

CC BY-NC

no

2022-01-12 14:27:35

Nucleic Acids Res. 2006 May 31; 34(10):3030-3043

PMC1474060

16738141

gkl378f3

Figure 3 Pes1 mutants M1 and M5 inhibit cell proliferation and elicit a reversible cell cycle arrest. ( A ) Equal numbers of TGR-1 cells stably transfected with the indicated constructs were seeded in multiples in the presence of 1 μg/ml doxycycline. Cells were trypsinized and counted by trypan blue exclusion after 6d. The histogram depicts the cell counts relative to the mock cell line after 6d. Error bars indicate SD. ( B ) Reversible cell cycle arrest by overexpression of mutants M1 and M5. Stably transfected TGR-1 cell lines were seeded at low density and treated with 1 μg/ml doxycycline for 30 h to induce expression of the specified constructs. Subsequently, the cells were incubated with 100 μM BrdU for 72 h to label proliferating cells. Visible light irradiation in the presence of Hoechst 33 258 selectively kills cells that have incorporated BrdU in their DNA. Arrested cells survive BrdU light treatment and give rise to colonies after withdrawal of doxycycline. Images show representative BrdU light assays conducted with the indicated cell lines.

CC BY-NC

no

2022-01-12 14:27:35

Nucleic Acids Res. 2006 May 31; 34(10):3030-3043

PMC1474060

16738141

gkl378f4

Figure 4 Dominant-negative Pes1 mutants inhibit pre-rRNA processing. ( A ) Schematic of eukaryotic pre-rRNA processing. The top diagram shows the primary 47S transcript at the correct scale and the position of the probes used in the northern blots. The lower diagram shows the processing of the pre-rRNA schematically. The effects of M1 and M5 overexpression are indicated. ( B ) Northern blot for pre-rRNA probed with ITS-2. Total RNA was extracted from subconfluent TGR-1 cells transfected with the indicated constructs after 30 h of induction. Hybridization with a probe against the 18S rRNA confirms equal loading of the blot. ( C ) Expression of dominant-negative Pes1 mutants impairs formation of mature 28S rRNA. Asynchronously growing TGR-1 cells were treated with doxycycline for 24 h. Cells were pulse labelled with 32 P-orthophosphate for 1 h and chased in regular medium for 0.5 and 3 h. ( D ) Ratio of 32S/28S rRNA at 3 h after metabolic labelling with 32 P-orthophosphate as described in (C).

CC BY-NC

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2022-01-12 14:27:35

Nucleic Acids Res. 2006 May 31; 34(10):3030-3043

PMC1474060

16738141

gkl378f5

Figure 5 Endogenous Pes1 is required for rRNA processing. ( A ) U2OS cells were transfected at day 0, 1 and 2 with either control or Pes1-specific siRNA. Endogenous Pes1 levels were analysed by western blot analysis 2d after the last transfection. Tubulin is shown as a loading control. ( B ) U2OS cells were transfected only twice at day 0 and 1 and metabolically labelled with 32 P-orthophosphate for 60 min at day 3. Subsequently, cells were incubated for 3 h in regular culture medium. Labelled rRNAs are indicated.

CC BY-NC

no

2022-01-12 14:27:35

Nucleic Acids Res. 2006 May 31; 34(10):3030-3043

PMC1474060

16738141

gkl378f6

Figure 6 Dominant-negative Pes1 mutants induce p53 accumulation in proliferating cells but not in quiescent cells. ( A ) Western blot for endogenous p53 levels. Stably transfected TGR-1 cells were induced at subconfluent density for 30 h with doxycycline. Cell lysates were prepared and analysed by immunoblotting for p53 accumulation using anti-p53 antibodies (Pab-240). n.s.: non specific. ( B ) Upper panel: western blot as described in (A), but the cells had been serum-starved for 72 h before induction. Lower panel: immunoblot against the HA-tag to confirm the expression of recombinant wt and mutant Pes1. ( C ) Analysis of the endogenous p53 response to expression of Pes1 wt, and mutants M1 and M5 by indirect immunofluorescence. Asynchronously proliferating and serum-starved (72 h) cells were treated with doxycycline for 24 h to induce the Pes1 constructs. Cells were fixed in methanol and stained against p53 with anti-p53 (Pab122). Cell nuclei were counterstained with DAPI. Representative images are shown. ( D ) Percentage of p53-positive nuclei in TGR-1 cells upon induction of Pes1 wt, and the dominant-negative mutants M1 and M5. Proliferating and serum-starved cells were analysed by immunofluorescence as described in (C). Numbers of examined cells are indicated in brackets. Error bars indicate SD of the percentage of p53-positive nuclei cells.

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2022-01-12 14:27:35

Nucleic Acids Res. 2006 May 31; 34(10):3030-3043

PMC1474060

16738141

gkl378f7

Figure 7 Dominant-negative mutants M1 and M5 associate with large pre-ribosomal particles. Western-blot analysis of sucrose gradient fractions of TGR-1 cells expressing Pes1-HA, and Pes1-HA mutants M1, M3 and M5 with an HA-specific antibody. RNA of each fraction was analysed by northern analysis with 28S rRNA specific probe (lower panel).

CC BY-NC

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2022-01-12 14:27:35

Nucleic Acids Res. 2006 May 31; 34(10):3030-3043

PMC1474060

16738141

gkl378f8

Figure 8 Incorporation of Pes1 mutants into the PeBoW-complex. U2OS cells stably transfected with the indicated constructs (A–J) were seeded at subconfluent density and treated with doxycycline for 20 h. The lysates were subjected to immunoprecipitation with antibodies either against the HA-tag (3F10), WDR12 (1B8), Bop1 (6H11) or the isotype control coupled to protein G Sepharose beads. The amount of protein loaded in each lane resembles an equivalent amount of total lysate or 20% thereof for the input. * indicates cross-reactivity to Ig molecules.

CC BY-NC

no

2022-01-12 14:27:35

Nucleic Acids Res. 2006 May 31; 34(10):3030-3043

PMC1474060

16738141

gkl378f9

Figure 9 Boundaries and domains important for Pes1 function and PeBoW formation. Pes1 deletions mutants of this work were compared with transposon insertion mutants published by Lapik et al . ( 15 ). Deletion mutants conferring a dominant-negative phenotype or affecting formation of PeBoW are indicated.

CC BY-NC

no

2022-01-12 14:27:35

Nucleic Acids Res. 2006 May 31; 34(10):3030-3043

PMC1474061

16738137

gkl373f1

Figure 1 Secondary structure models of the HIV-2 TAR RNA. The models represent the lowest energy solution obtained using the Mfold algorithm and are supported by experimental data. The previously reported branched hairpins form ( A ) stay in equilibrium with the new extended hairpin form represented by two conformers E1 and E2 ( B ). The hairpins I and II of the branched form constitute the arms of the main hairpin of the extended form. Consequently, the branch site between two hairpins becomes an apical loop. The small hairpin, coded III in the branched hairpins structure is identical for all conformers.

CC BY-NC

no

2022-01-12 14:27:35

Nucleic Acids Res. 2006 May 31; 34(10):2984-2997

PMC1474061

16738137

gkl373f2

Figure 2 Magnesium-dependent mobility of TAR-2 RNA and TAR-2 A21 mutant on non-denaturing PAGE. (A) 123 nt TAR-2 wt and its A21 mutant. ( B ) Plotting of the phoshoimager quantification of the TAR-2 wt fast and slow migrating bands ratio, averaged from several experiments. Alternatively folded monomeric RNAs are indicated as M and the dimers as D. Lanes F correspond to formamide-denatured control samples. MgCl 2 concentrations (mM) are indicated above the respective lanes. The samples were analysed on a 6% non-denaturing 0.5× TB with 0.1% Triton X-100 gels. Similar results were obtained in 0.5× TBE buffer without Triton X-100 (Figure S1A).

CC BY-NC

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2022-01-12 14:27:35

Nucleic Acids Res. 2006 May 31; 34(10):2984-2997

PMC1474061

16738137

gkl373f3

Figure 3 Secondary structure probing of HIV-2 TAR RNA. The RNA was treated with selected single-strand specific enzymes (S1, T1, A), DEPC and double-strand specific RNase V1. ( A ) Cleavage patterns obtained for the 5′ end labelled TAR-2 wt transcript. Lanes C represent control sample with untreated RNA; lanes L, formamide ladder; lanes T, limited hydrolysis with RNase T1. ( B ) Cleavage patterns obtained for the 5′ end labelled TAR-2 A21 mutant transcript. ( C ) A summary of the enzymatic cleavages and chemical modifications data for the TAR-2 wt RNA viewed on the secondary structure models (E1, E2 and B). For clarity, only the top part of the E2 conformer that differs from E1 is shown. Sites and intensities of cleavages with the respective reagents are indicated by symbols (see inset); the size of a symbol corresponds to the relative cleavage intensity. The weakest cleavages are not indicated. ( D ) Two different RNase T1 cleavage patterns observed for the particular G-rich region of both TAR-2 wt and A21 mutant along with respective secondary structure motifs. Both patterns point to the mixture of TAR-2 forms; the right pattern is consistent mostly with one of the extended (E1) and with the branched (B) structure models; the most often observed left pattern represents predominantly the second extended conformer E2. ( E ) RNase T1 cleavage pattern obtained for the TAR-2 B4 and ΔC23 mutants stabilized in the branched (B) form.

CC BY-NC

no

2022-01-12 14:27:35

Nucleic Acids Res. 2006 May 31; 34(10):2984-2997

PMC1474061

16738137

gkl373f4

Figure 4 Metal ions-induced TAR-2 RNA structure probing. ( A ) Pb 2+ -induced RNA cleavage of TAR-2 wt and TAR-2 A21 mutant. ( B ) Mg 2+ -induced RNA cleavage of TAR-2 wt and TAR-2 A21 mutant. Lane C represents control sample with untreated RNA; lane L, formamide ladder; lane T, limited hydrolysis with RNase T1. ( C ) A summary of the Pb 2+ - and Mg 2+ -ions induced RNA cleavages of the TAR-2 wt, viewed on the secondary structure models (B, E1, E2). For clarity, only the top part of the E2 conformer that differs from E1 is shown. Sites and intensities of cleavages are indicated by arrows (see insert); size of symbols corresponds to the relative cleavage intensity. The weakest cleavages are not indicated. ( D ) The comparison of the inhibitory effect of Mg 2+ on Pb 2+ -induced cleavages at U27 position of TAR-2 wt, TAR-2 A21 mutant and TAR-2 isolated hairpin I.

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2022-01-12 14:27:35

Nucleic Acids Res. 2006 May 31; 34(10):2984-2997

PMC1474061

16738137

gkl373f5

Figure 5 Gel shift assays of the Tat-2 binding to: ( A ) TAR-2 wt and its mutants, ( B ) isolated hairpins I and II corresponding to the TAR-2 RNA branched structure. ( C ) Panel C shows the plot of the Tat-2 binding affinity observed for the TAR-2 wt, TAR-2 mutants and isolated hairpins I and II. Amounts of the Tat-2 protein (ng per reaction) are indicated below their respective lanes.

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2022-01-12 14:27:35

Nucleic Acids Res. 2006 May 31; 34(10):2984-2997

PMC1474061

16738137

gkl373f6

Figure 6 Inhibitory effect of argininamide on Pb 2+ -induced cleavages. ( A ) Gel patterns are shown for TAR-2 wt, TAR-2 A21 mutant, hairpin I, hairpin W [used in the previous NMR studies, Refs ( 28 , 29 )] and hairpin II. 2D structures of respective hairpins are presented below gels with cleavage position indicated. Lane L represents formamide ladder; lane T, limited hydrolysis with RNase T1. ( B ) Shows the plot of the argininamide inhibitory effect on the Pb 2+ -induced cleavages at U27 (U23, as in original report, for the hairpin W) or U62 residues of analysed RNAs.

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no

2022-01-12 14:27:35

Nucleic Acids Res. 2006 May 31; 34(10):2984-2997

PMC1474062

16738142

gkl397f1

Figure 1 Sequences of DNA used in the AFM experiments. ( a ) Schematic representation of the PPC (2885) and sequence of the region analyzed (953 bp) that contains the hyperperiodic fragment Ω4 from C.elegans (923 bp). In the PPC the hyperperiodic region is located at both ends of the molecule, hence tracing of the molecule is independent of the particular end chosen as a starting point. Capital case letters denote the hyperperiodic fragment Ω4 from C.elegans . ( b ) Sequence of SQ100 fragment (185 bp, non-periodic) that contains the fragment Ω7 and ( c ) sequence of SQ322 (182 bp, hyperperiodic) that contains the fragment Ω6 from C.elegans . Two or more consecutive As or Ts were colored in green and red, respectively. The sequences were plotted in columns of 10 bases to highlight ∼10 bp periodicities. Capital letters in (a) denote the hyperperiodic fragment Ω4 from C.elegans and in (b and c) denote the Ω7 and Ω6 fragments, respectively.

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2022-01-12 14:27:35

Nucleic Acids Res. 2006 May 31; 34(10):3057-3066

PMC1474062

16738142

gkl397f2

Figure 2 Migration of hyperperiodic and control DNA in agarose gels. ( a ) Two-dimensional 4% NuSieve agarose gel electrophoresis of lambda DNA fragments (cut with NciI and PvuII) and ( b ) lambda fragments plus Ω4 fragments cut with the same restriction enzymes. Gels were run first at 4°C in the absence of intercalator (Ethidium Bromide, vertical) and then at 37°C in the presence of intercalator in the perpendicular direction (horizontal). The hyperperiodic Ω4 fragments (indicated by arrows) migrated more slowly than one would expect on the basis of its length. Similar anomalies were observed when using 2% agarose gels (data not shown). Gel electrophoresis on fragments SQ100 (185 bp, non-periodic, lane 3) and SQ322 (182 bp, hyperperiodic, lane 4) run at 37°C with EtBr ( c ) and at 4°C without EtBr ( d ). Lanes 1, 2, 5 and 6 correspond to 100 and 25 bp DNA ladders. At low temperature, the hyperperiodic fragment migrates more slowly than expected.

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2022-01-12 14:27:35

Nucleic Acids Res. 2006 May 31; 34(10):3057-3066

PMC1474062

16738142

gkl397f3

Figure 3 High-resolution AFM images of DNA samples. ( a ) Two representative DNA molecules of the PPC adsorbed on a mica surface. ( b ) Illustration of the data analysis and tracing of the DNA molecules. A segmented line made of points separated by l = 2.5 nm follows the contour of the molecule. The important parameters we employ are the contour length spacing L , the end-to-end distance R and the curvature C defined as the inverse of the curvature radius r that embraces three contour points, C = 1 /r . In the inset in ( b ), these parameters are shown together with an example of three values of R for the same contour length spacing L , in this case L = 3 × l = 7.5 nm. We also show an example for the calculation of r for three points separated by L = 2 × l = 5 nm (red arc). ( c ) AFM image of sample SQ100 and histogram of contour lengths L 0 [inset in ( c )]. ( d ) AFM image of sample SQ322 and histogram of contour lengths L 0 [inset in (d)].

CC BY-NC

no

2022-01-12 14:27:35

Nucleic Acids Res. 2006 May 31; 34(10):3057-3066

PMC1474062

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gkl397f4

Figure 4 Analysis of unusual character in hyperperiodic DNA from C.elegans using AFM. ( a and b ) Plots of < R 2 > versus contour length spacing L of the ends of the PPC, control DNA, SQ100 and SQ322 samples. Different sets of data in ( a ) correspond to different experiments, mica surfaces and sample preparations. In total, 190 PPC fragments were studied. The control DNA follows the prediction given by the worm-like-chain model with a persistence length of 51 nm. The hyperperiodic DNA regions from the C.elegans genome clearly show a very different behavior, characterized by lower < R 2 >. To guide the eye, predictions from WLC with different values for the persistence length are included (solid grey). The five curves included are for P = 20, 30, 40, 50 and 60 nm. The slope of the curves increases as P increases. Bending of the DNA, is evidenced by lower < R 2 > than predicted by WLC. ( c ) Normalized histograms of absolute curvature | C | for the PPC and the control DNA sample for L = 5 nm and for L = 25 nm [inset in (c)]. ( d ) Normalized absolute curvature | C | histograms for SQ100 and SQ322 for L = 5 nm. Distributions were fit to a Gaussian function centered at zero. The hyperperiodic DNA consistently showed larger standard deviations, independently of the spacing L [inset in (c)]. Bins and associated errors (square root of number of points in the bin) were normalized by the area of the histogram. We note that occasionally one cannot distinguish the error bars because they are smaller than the points.

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Nucleic Acids Res. 2006 May 31; 34(10):3057-3066

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Figure 5 Measurement of the intrinsic curvature in hyperperiodic C.elegans DNA. ( a ) Curvature correlation function ( Equation 2 in the main text) for PPC and Control DNA calculated for curvatures measured at contour length separations of L = 5, 15 and 25 nm. A long-range persistence of handedness in hyperperiodic DNA is evident from the graph, indicating the presence of intrinsic bending. ( b ) Normalized histograms of absolute mean curvatures per molecule |< C > molecule | for the PPC and the control DNA sample for L = 5 nm and for L = 25 nm [inset in (b)]. ( c ) |< C > molecule | histograms for SQ100 and SQ322 for L = 5 nm. Hyperperiodic DNA showed a preferred mean curvature per molecule of ∼0.01 nm −1 at L = 5 nm, consistent with the presence of intrinsic bending. Bins and associated errors (square root of number of points in the bin) were normalized by the area of the histogram.

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2022-01-12 14:27:35

Nucleic Acids Res. 2006 May 31; 34(10):3057-3066

PMC1474063

16738131

gkl374f1

Figure 1 Depletion of SMN in Hela cells using an RNAi approach ( A ) western blot analysis on SMN-depleted cells. An siRNA duplex has been transfected into Hela cells for the indicated time, and extracts were prepared and examined for SMN levels by western blotting using anti-SMN antibodies. A control siRNA was introduced into HeLa cells under similar conditions. The GAPDH protein was used as a loading control. ( B ) Quantification of SMN protein amounts. Blots were scanned and quantified using ImageQuant software (Molecular Dynamics). The percentage of SMN levels with respect to control is shown. ( C ) Immunofluorescence studies on SMN-depleted and control Hela cells using anti-SMN antibodies. In control cells, the SMN protein localizes to the cytoplasm and to Cajal bodies, while cytoplasmic and Cajal bodies fluorescence are no longer visible in the SMN-depleted cells. The nucleus was stained with DAPI. Note that the cell presenting Cajal bodies staining in the SMN-depleted cells (right panel) has not taken up the siRNA and can be considered as a control cell.

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Nucleic Acids Res. 2006 May 31; 34(10):2925-2932

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Figure 2 Glycerol gradient sedimentation of snRNPs. Extracts prepared from SMN-depleted (siRNA-SMN) and control cells (control) after 60 h were analyzed by glycerol gradient centrifugation and fractions were recovered. The RNA was extracted, run on denaturing polyacrylamide gels and transferred to a nylon membrane for northern analysis using 32 P-labelled oligonucleotide probes specific for U1, U2, U4, U5 and U6 snRNAs. The fraction number and the positions of the different snRNP complexes are shown at the bottom.

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Nucleic Acids Res. 2006 May 31; 34(10):2925-2932

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Figure 3 Depletion of SMN hinders the efficient nuclear import of a transiently expressed GFP-SmB fusion protein. After transfection of Hela cells with a siRNA duplex specific to SMN or a control siRNA for 36 h, cells were transfected with a plasmid encoding a GFP-SmB gene. After 24 h, cells were fixed and the subcellular localization of the GFP-SmB fusion protein was then observed by fluorescence microscopy. Cajal bodies were visualized by immunofluorescence using anti-coilin antibodies. As shown in the upper panels, in control cells, the GFP fusion protein localizes to the nuclear compartment and in Cajal bodies while in the SMN-depleted cells (lower panels), a cytoplasmic accumulation of the GFB-SmB fusion protein is observed.

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Nucleic Acids Res. 2006 May 31; 34(10):2925-2932

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Figure 4 SMN-depleted cells contain numerous foci reacting with coilin antibodies. ( A ) Immunofluorescence studies with antibodies directed against coilin and SMN were performed on control cells and on SMN-depleted cells. In control cells, coilin and SMN localizes to canonical Cajal bodies while these structures become dispersed in SMN-depleted cells where coilin localizes to numerous nucleoplasmic foci. Zooms of representative cells corresponding to the insets are represented at the right. ( B ) Relocalization of coilin into nucleoli upon SMN depletion. Immunofluorescence studies with coilin allows the detection of Cajal bodies in control cells while fibrillarin is found primarily in nucleoli and also in Cajal bodies (white arrows, panel b). In SMN-depleted cells (lower panels), coilin is found in residual Cajal bodies and in nucleoli (white arrows, panel h) which are stained by fibrillarin (panels f–g). The nucleus was stained with DAPI.

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Nucleic Acids Res. 2006 May 31; 34(10):2925-2932

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Figure 5 snRNPs do not localize in residual Cajal bodies observed in SMN-depleted cells. To determine the localization of snRNPs, control cells and SMN-depleted cells were subjected to immunofluorescence studies using anti-p80 coilin (red) and anti-m 3 G antibodies (green). In control cells, snRNPs accumulate into Cajal bodies and into speckles while in SMN-depleted cells, snRNPs are not located into residual Cajal bodies but are found in a diffuse speckled distribution.

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Nucleic Acids Res. 2006 May 31; 34(10):2925-2932

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Figure 6 In situ localization of U85 scaRNA. A plasmid containing human U85 scaRNA was transfected into HeLa cells with control or SMN specific siRNA duplexes and after 48 h, cells were subjected to in situ hybridization using a Cy3-fluorescent probe specific to U85 (red). The canonical Cajal bodies in control cells and the residual Cajal bodies in SMN-depleted cells were visualized with anti-p80 coilin antibody staining (green).

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2022-01-12 14:27:35

Nucleic Acids Res. 2006 May 31; 34(10):2925-2932

PMC1474065

16738132

gkl383f1

Figure 1 Trypsin cleavage of WT and mutant TRAP proteins. TRAP proteins were digested with 5% trypsin either in the absence (lanes 1–4) or presence (lanes 5–8) of tryptophan and samples were removed at various times. Cleavage was monitored on 16% SDS–polyacrylamide gels that were stained with GelCode Blue (Pierce). Bands were quantitated using ImageQuant (Molecular Diagnostic) and the results were plotted as percentage cleavage versus incubation time with trypsin.

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Nucleic Acids Res. 2006 May 31; 34(10):2933-2942

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Figure 2 Ribbon diagram of TRAP complexed with RNA. One complete subunit (light blue) and parts of the two adjacent subunits (green and brown) in TRAP are shown. The loop formed from residues 25–33 and the adjacent β-strand (residues 34–38) proposed to be key in tryptophan-mediated activation of RNA binding are highlighted in dark blue. The β-strands are depicted as arrows and the bound tryptophans as ball and stick models. The side chains of several key amino acids are shown in either ball and stick models on the complete subunit. RNA binding residues are in red, Thr30 is in magenta. The RNA is shown as golden stick diagrams and the bases from one GAGAU repeat are labeled. The hydrogen bonds between Thr30 and the bound tryptophan as well as to the third G are shown as dashed lines.

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2022-01-12 14:27:35

Nucleic Acids Res. 2006 May 31; 34(10):2933-2942

PMC1474067

16738135

gkl357f1

Figure 1 Detection of 28S and 18S rRNA transcripts containing homopolymeric and heteropolymeric poly(A)-tails using oligo(dT) primed RT–PCR. The 28S and 18S rRNAs are schematically presented. The gene specific forward primers used for the PCR amplification and screening of the oligo(dT)-primed cDNAs are shown as arrows below the gene. Thin vertical lines indicate the positions of the added poly(A)-tail of each clone and the tail compositions are shown. The exact positions of the polyadenylated nucleotides, according to the gene sequence, are presented in the Supplementary Tables S3 and S4.

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Nucleic Acids Res. 2006 May 31; 34(10):2966-2975

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Figure 2 Detection of ESTs related to polyadenylated 28S and 18S rRNA transcripts. ESTs containing the 28S or 18S rRNAs with poly(A) or poly(A)-rich tails were identified by the bioinformatic tool, PolyAfinder. The points of addition of the poly(A) or poly(A)-rich tails are presented as thin vertical lines, while the length of the line indicates the number of ESTs which were polyadenylated at this location. Examples of the sequences of several heteropolymeric tails are presented in Table 1 and a full list, including the polyadenylation positions according to the gene sequence as well, is presented in Supplementary Tables S1 and S2 of the supplementary Data.

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Nucleic Acids Res. 2006 May 31; 34(10):2966-2975

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Figure 3 PE analysis of the 28S rRNA. ( A ) In an attempt to detect distal cleavage products, total RNA isolated from the cell types indicated at the top of lanes 5–8 was annealed to [ 32 P] primer 28Sp1 and extended with reverse transcriptase. The resulting cDNA was fractionated on 7% denaturing PAGE along with a DNA sequencing ladder generated using a 28S rRNA DNA template and the same primer (lanes 1–4). The nucleotide numbers of the sequence of 28S rRNA are indicated to the left. In order to estimate the amount of the putative truncated transcripts, the mature 5′ end of the molecule was detected by PE with primer 28Sp5, located 99 nt downstream to the mature 28S rRNA 5′ end, using RNA purified from CCRF-CEM cells. Appropriated dilutions of this reaction, as shown at the top of lanes 9–12 were fractionated on the same gel. ( B ) Control reactions, in which RNA purified from CCRF-CEM cells was analyzed by PE in the presence of dNTPs at concentrations of 1 or 0.004 mM (lanes 1 and 2, respectively) in order to distinguish whether the observed signals were caused by modified nucleotides, are shown. Note that the significant signal located at nucleotide 1733 was not detected in this experiment. In lane 3, in vitro transcribed RNA corresponding to the analyzed region of 28S rRNA, was analyzed by PE to ensure that the signals did not arise from secondary structures that inhibit the reverse transcriptase.

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Nucleic Acids Res. 2006 May 31; 34(10):2966-2975

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Figure 4 cRT–PCR analysis detected additional 28S fragments which terminate in the region disclosed by oligo(dT) RT–PCR and PE. The combined results of the analysis of the 28S rRNA for truncated and polyadenylated fragments using cRT–PCR, oligo(dT) RT–PCR (dT-RT–PCR) and PE are schematically presented along the region of 1250–2100 nt. Horizontal lines represent the cDNAs obtained by each technique and are drawn to scale. The primers used for the PCR amplification and screening of the dT-RT–PCR and cRT–PCR cDNA, as well as the PE are shown by arrows. Details about the cRT–PCR clones are displayed in the Supplementary Table S5 and the clone # in the figure is related to that which appears in this table. The defined region of 1670 to 1780 that is enriched with 5′ and 3′ ends of detectable cleavage products is indicated with a gray background. Inset: A diagram explaining the experimental design. Following cleavage, the 28S rRNA is separated to proximal (upstream) and distal (downstream) fragments. PE discloses the 5′ end of the distal fragment while cRT–PCR and oligo(dT) RT–PCR analysis reveal the 3′ end of the proximal fragment, to which the poly(A) tail is added. Arrow heads indicate the primers used and dashed lines the cDNAs produced.

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2022-01-12 14:27:35

Nucleic Acids Res. 2006 May 31; 34(10):2966-2975

PMC1475745

16757577

gkl304f1

Figure 1 Induction of ER stress genes. Semi-quantitative RT–PCR analysis of ER stress induced genes in HepG2 cells treated for the indicated hours with thapsigargin (Tg). A control 24 h treatment with DMSO is shown on the right. The PCR cycles are shown for each gene, to indicate the relative abundance of the mRNAs analyzed. Quantification of the RT–PCRs are shown in appropriate panels. The colors refer to the different time points analyzed.

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Nucleic Acids Res. 2006 Jun 6; 34(10):3116-3127

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gkl304f2

Figure 2 Recruitment of GTFs. Kinetic ChIP analysis of Pol II, TBP and p300 recruitment on representative ER stress promoters in HepG2 cells. Several semi-quantitative PCRs were analyzed, scanned and the data plotted against the negative control (Flag antibody). Values are represented as fold-enrichment over the Flag control. The bars of the standard deviations result from the quantification of several PCRs within the linear range. Note the different scales in some of the panels. The basal, uninduced levels are in white, 1 h in light green, 4 h in green and 8 h in dark green.

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Nucleic Acids Res. 2006 Jun 6; 34(10):3116-3127

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Figure 3 Changes in histones acetylation and methylation upon ER stress. Kinetic ChIP analysis of HepG2 cells as in Figure 2 , with antibodies directed anti-AcH4, anti-AcH3, H3-K4-me2, H3-K4-me3, H3-K79-me2 and unmodified H3. The results shown are expressed as enrichment over the negative control antibody.

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Nucleic Acids Res. 2006 Jun 6; 34(10):3116-3127

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Figure 4 H3 recovery in ChIP assays under ER stress. ( A ) Kinetic ChIP analysis with the indicated antibodies against unmodified H3, H3-K9-me2 and H3-K27-me3 in HepG2 cells, treated for the indicated times with Tg. The different targets were PCR-amplified with specific primers. A control satellite centromeric 11 region was added in the upper left panel. ( B ) Same as (A) on different regions of the CHOP gene, a scheme of which is depicted.

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Nucleic Acids Res. 2006 Jun 6; 34(10):3116-3127

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Figure 5 Binding of ER stress induced TFs to ER stress promoters. Kinetic ChIP analysis of XBP-1, CHOP, ATF6 and C/EBPβ to ER stress promoters in HepG2 cells. Chromatin was prepared as in Figures 2 – 4 under growing and 1, 4 and 8 h post ER stress induction.

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Nucleic Acids Res. 2006 Jun 6; 34(10):3116-3127

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Figure 6 Binding of NF-Y, Sp1 and p53 to ER stress promoters. ChIP analysis of NF-Y, Sp1 and p53 in HepG2 cells. Chromatin was prepared from cells treated for the indicated times with Tg. Two semi-quantitative PCR amplifications are shown for most targets. The number of PCR cycles are shown on the left.

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2022-01-12 14:27:35

Nucleic Acids Res. 2006 Jun 6; 34(10):3116-3127

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Figure 7 NF-YB inactivation: induction of ER stress genes. HCT116 cells were transfected with an siRNA oligonucleotide that targets the NF-YB subunit of NF-Y. ( A ) Western blot analysis NF-YB (and of the control Vinculin) with extracts from uninduced and ER stress induced cells. ( B ) RT–PCR analysis of ER stress response genes in HCT116 cells treated as in (A). In the lower panel, the analysis of genes with CCAAT-less promoters are shown. ( C ) ChIP analysis of HCT116 cells in uninduced conditions with anti-YB antibodies. The indicated ER stress promoters were analyzed by semi-quantitative PCR analysis.

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2022-01-12 14:27:35

Nucleic Acids Res. 2006 Jun 6; 34(10):3116-3127

PMC1475746

16757580

gkl396f1

Figure 1 Splice graph of a three-exon alternatively spliced gene. ( a ) Gene structure for a three-exon gene. The second exon is a cassette exon. ( b ) The splice graph representation of the gene structure. The exon skipping event is represented by a directed edge from node 1 to node 3.

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2022-01-12 14:27:35

Nucleic Acids Res. 2006 Jun 6; 34(10):3150-3160

PMC1475746

16757580

gkl396f2a

Figure 2 Probabilistic isoform reconstruction for a simulated gene. Left panel: sequence observations; right panel: results of probabilistic isoform reconstruction. The upper graph indicates the overall log likelihood; the lower graph shows the estimated probabilities for individual isoforms until convergence. A – D represents different situations of sequence observations.

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Nucleic Acids Res. 2006 Jun 6; 34(10):3150-3160

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gkl396f3

Figure 3 Simulation study to test the robustness of the EM algorithm. X-axis: the total number of sequence observations being simulated; Y-axis: the total variation distance between the true probabilities and the estimated probabilities (see Materials and Methods). ( A ) Simulation studies using fixed probabilities of four isoforms. The probabilities are listed in Table 1 . ( B ) A simulation study using randomized probabilities of four isoforms.

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Nucleic Acids Res. 2006 Jun 6; 34(10):3150-3160

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Figure 4 Probabilistic isoform reconstruction for HLA-DMB. ( A ) Gene structure and alternative splicing of HLA-DMB. Exon 4 encodes the TM domain. Exon 5 encodes the LT signal. ( B ) Four putative isoforms of HLA-DMB. ( C ) Probabilistic isoform reconstruction of HLA-DMB. The upper graph indicates the overall log likelihood; the lower graph shows the estimated probabilities for individual isoforms until convergence. The FL form has the highest estimated probability, followed by Δ-LT, Δ-TM and Δ-TM,LT. ( D ) RT–PCR analysis of HLA-DMB isoforms in pooled human tissues (see Appendix 3 of the online supplements for details of the experiment). Left lane: marker; Right lane: HLA-DMB.

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Nucleic Acids Res. 2006 Jun 6; 34(10):3150-3160

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Figure 5 Probabilistic isoform reconstruction for TPM1. ( A ) The gene structure of TPM1 from exon 4 to exon 11. ( B ) EST evidence indicates coupled alternative splicing events in TPM1. ( C ) Probabilistic isoform reconstruction of TPM1. The upper graph indicates the overall log likelihood; the lower graph shows the estimated probabilities for individual isoforms until convergence. Only isoforms with >0.05 probability are shown in the graph.

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Nucleic Acids Res. 2006 Jun 6; 34(10):3150-3160

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Figure 6 CPU time of probabilistic isoform reconstruction for 186 genes on human chromosome 22. X-axis: numbers of putative isoforms. Y-axis: CPU time on a PC (AMD Athlon 1500+ with 320MB of RAM).

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Nucleic Acids Res. 2006 Jun 6; 34(10):3150-3160

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gkl381f1

Figure 1 Involvement of candidate T2D genes in cellular metabolism pathways, selected candidates are shown in red.

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2022-01-12 14:27:35

Nucleic Acids Res. 2006 Jun 6; 34(10):3067-3081

PMC1475749

16757573

gkl386f1

Figure 1 Heat maps; effects of NO • and the p38 MAPK inhibitor SB202190 (SB) on mRNA degradation as determined by microarray. THP-1 cells (2 × 10 7 ) were stimulated with LPS (1 μg/ml) for 4 h. After 30 min treatment with ActD (2.5 μg/ml) in the absence or presence of SB (0.1 μM), cells were incubated with GSNO (400 μM) or control GSH (400 μM) for 0–180 min. At the indicated time-points, cells were harvested to extract total RNA for microarray analysis. The half-lives of 220 genes were found to be differentially regulated (see Materials and Methods). ( A ) Hierarchical clustering of normalized mean signal intensities from four independent experiments for all 220 genes at each time point and condition. ( B ) Same results as (A) after conversion of individual time point data into slopes based on a first order mRNA decay model.

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Nucleic Acids Res. 2006 Jun 6; 34(10):3044-3056

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Figure 2 Effects of NO • and the p38 MAPK inhibitor SB202190 (SB) on MAPK phosphorylation. ( A ) NO • increases p38 MAPK phosphorylation, an effect blocked by SB (0.1 μM). ( B ) NO • increases Erk1/2 phosphorylation, an effect enhanced by SB (0.1 μM). ( C ) SB (0–5 μM) alone increases Erk1/2 phosphorylation. THP-1 cells (1 × 10 7 ) were stimulated with LPS (1 μg/ml) for 4 h. After 30 min treatment with ActD (2.5 μg/ml) in the absence (control) or presence of SB, cells were incubated without or with GSNO (0–800 μM) for another 30 min, as indicated and then lysed. Each experiment was repeated at least twice with similar results.

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Nucleic Acids Res. 2006 Jun 6; 34(10):3044-3056

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Figure 3 NO • stabilizes ( A ) MAP3K7IP2, ( B ) MRPS18A and ( C ) TP53BP2 mRNA through Erk1/2 as determined by RT–PCR. Left panels show the effects of NO • and the p38 MAPK inhibitor, SB202190 (SB; 0.1 μM), on mRNA degradation. Right panels show the effects of the Erk1/2 inhibitor, PD98059 (PD; 30 μM), on mRNA degradation in the presence of SB. THP-1 cells (2 × 10 7 ) were stimulated with LPS (1 μg/ml) for 4 h. After 30 min treatment with transcription inhibitor ActD (2.5 μg/ml) in the absence or presence of indicated MAPK inhibitors, cells were incubated with GSNO (400 μM) or GSH control (400 μM) for 0–180 min. All mRNA levels were quantitated by TaqMan ® RT–PCR and normalized to GADPH mRNA. Data, presented as percentage relative to mRNA levels at 0 min, are the mean ± SEM of three independent experiments. The respective mRNA half-lives of MAP3K7IP2, MRPS18A and TP53BP2 were as follows: 179, 98 and 91 min for control GSH; 236, 132 and 121 min for GSNO; 200, 103 and 119 min for SB/GSH; 314, 166 and 155 min for SB/GSNO; 171, 89 and 100 min for SB/PD/GSH; and 160, 90 and 103 min for SB/PD/GSNO.

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2022-01-12 14:27:35

Nucleic Acids Res. 2006 Jun 6; 34(10):3044-3056

PMC1475749

16757573

gkl386f4

Figure 4 NO • stabilizes CURE-containing mRNA but inhibits its translation through an Erk1/2-dependent mechanism. ( A ) Effect of the Erk1/2 inhibitor, PD98059 (PD; 30 μM), on LUC mRNA levels and LUC activity, respectively. THP-1 cells, transfected with pGL3/CURE, mutant pGL3/CUREmut or control pGL3, were treated with ActD (2.5 μg/ml) for 30 min (for mRNA determinations only) and then incubated with GSH (400 μM) or GSNO (400 μM) for 5 h to measure LUC mRNA by TaqMan ® RT–PCR or for 20 h to measure LUC activity. ( B ) Effect of a Mek1 dominant-negative mutant on LUC mRNA levels and LUC activity, respectively. THP-1 cells, co-transfected with pGL3/CURE or mutant pGL3/CUREmut or control pGL3 plus either pUSEamp (empty vector) or pMEK1-DN (dominant-negative Mek1), were similarly treated as in A for measurement of LUC mRNA levels and LUC activity. Data, presented as percentage relative to LUC mRNA level or LUC activity of pGL3, are the mean ± SEM of three to six independent experiments. ( C ) Effect of NO • on the expression of MAP3K7IP2, a naturally-occurring, CURE-containing gene. THP-1 cells (1 × 10 7 ) were pretreated with SB (0.1 μM) or PD (30 μM) for 30 min. After 20 h incubation of GSH (400 μM) or GSNO (400 μM), cells were then lysed for western blotting. Each experiment was repeated twice with similar results.

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Nucleic Acids Res. 2006 Jun 6; 34(10):3044-3056

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Figure 5 Role of hnRNP K and hnRNP E2/E1 in NO • -Erk1/2-CURE signaling. ( A ) RNA REMSAs with either a consensus (left panel) or MAP3K7IP2 (right panel) CURE riboprobes. GSNO (400 μM) treatment for 3 h increased complex formation compared to control GSH; anti-hnRNP K and anti-hnRNP E2/E1 both super-shift the complex; the unlabeled CURE riboprobes, but not the mutant of consensus CURE (mutant CURE) compete with the labeled CURE riboprobes. ( B ) Translocation of hnRNP K and hnRNP E2/E1 to the cytoplasm by western blotting. GSNO (400 μM) treatment for 3 h increased the presence of hnRNP K and hnRNP E2/E1 in the cytoplasm but not in whole-cell lysates compared to control GSH. This effect was further enhanced by the p38 MAPK inhibitor SB202190 (SB; 0.1 μM), but blocked by the Erk1/2 inhibitor PD98059 (PD; 30 μM). A control protein α tubulin is shown for comparison. Experiments in (A and B) were repeated at least twice with similar results. ( C ) Overexpression of hnRNP K mimicked the effect of NO • , repressing the expression of a chimeric LUC-CURE reporter gene. THP-1 cells were co-transfected with pGL3/CURE, pGL3/CUREmut or control pGL3 and pcDNA3 (empty vector) or phnRNP-K (hnRNP K expression plasmid). After treatment with GSH (400 μM) or GSNO (400 μM) for 20 h, LUC activities were measured. Data, presented as percentage relative to LUC activity with pGL3, are the mean ± SEM of three independent experiments.

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2022-01-12 14:27:35

Nucleic Acids Res. 2006 Jun 6; 34(10):3044-3056

PMC1475750

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gkl389f1

Figure 1 ERΔ5 represses ERα transcriptional activity through interaction with ERα. ( A ) ERΔ5 represses transcription of the ERE-Luc, pS2-Luc and C3-Luc reporters. HepG2 cells were co-transfected with 0.5 μg of the expression vector for ERΔ5, 0.5 μg of the expression plasmid for ERα and 0.2 μg of various luciferase reporter plasmids in the absence or presence of 10 nM of 17β-estradiol (E 2 ). The luciferase activity obtained on transfection of the respective luciferase reporter and ERα without exogenous ERΔ5 in the absence of E 2 was set as 1. ( B ) In vitro interaction of ERΔ5 with ERα. Glutathione–Sepharose beads bound with GST-ERα or with GST were incubated with 35 S-labeled ERΔ5 in the absence or presence of 100 nM E 2 . After washing the beads, the bound proteins were eluted and subjected to SDS–PAGE and autoradiography. ( C ) In vivo interaction of ERΔ5 with ERα. ERα and FLAG-tagged ERΔ5 were co-transfected into HepG2 cells in the presence or absence of 10 nM E 2 . Cell lysates were immunoprecipitated (IP) by anti-FLAG M2 monoclonal antibody (Sigma), and the precipitates were then immunoblotted (IB) with anti-ERα polyclonal antibody (Santa Cruz Biotech).

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2022-01-12 14:27:35

Nucleic Acids Res. 2006 Jun 6; 34(10):3095-3106

PMC1475750

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Figure 2 HBx inhibits ERα-mediated transactivation function in hepatoma cells. ( A ) HepG2 cells were co-transfected with 0.2 μg of ERE-Luc, 50 ng of the expression plasmid for ERα and increasing amounts of the expression plasmid for FLAG-tagged HBx in the absence or presence of 10 nM E 2 . The luciferase activity obtained on transfection of ERE-Luc and ERα without exogenous HBx in the absence of E 2 was set as 1. ( B ) Immunoblotting showing the ERα and HBx levels in HepG2 cells. Cells were transfected as in (A). Whole cell extracts were prepared from the cells transfected with 2.0 μg of the expression plasmid for HBx in the presence of 10 nM E 2 , and were detected with anti-ERα (Santa Cruz Biotech), anti-FLAG (Sigma) or anti-GAPDH (Biogenesis) antibody. (C–E) HepG2 cells were co-transfected with 50 ng of the expression plasmid for ERα, 1.0 μg of the expression plasmid for FLAG-tagged HBx, and 0.2 μg of C3-Luc ( C ), pS2-Luc ( D ) or pS2ΔERE-Luc ( E ), in the absence or presence of 10 nM E 2 . The luciferase activity obtained on transfection of the respective luciferase reporter without exogenous ERα and HBx in the absence of E 2 was set as 1.

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2022-01-12 14:27:35

Nucleic Acids Res. 2006 Jun 6; 34(10):3095-3106

PMC1475750

16757575

gkl389f3

Figure 3 HBx and ERΔ5 have additive effect on repression of specific ERα responsive gene transcription. (A–D) HepG2 cells were co-transfected with 50 ng of the expression plasmid for ERα, 1.0 μg of the expression plasmid for FLAG-tagged HBx, 50 ng of the expression plasmid for ERΔ5, and 0.2 μg of ERE-Luc ( A ), C3-Luc ( B ), pS2-Luc ( C ) or pS2ΔERE-Luc ( D ), in the absence or presence of 10 nM E 2 . The luciferase activity obtained on transfection of the respective luciferase reporter without exogenous ERα, ERΔ5 and HBx in the absence of E 2 was set as 1. ( E ) HepG2 cells were co-transfected with 0.2 μg of pS2-Luc, 50 ng of the expression plasmid for ERα, 1.0 μg of the expression plasmid for FLAG-tagged HBx and 50 ng of the expression vector for ERΔ5. Cells were then treated with control (0.1% ethanol) vehicle, 10 nM E 2 , 100 nM 4-hydroxytamoxifen (4-OHT) or 10 nM E 2 plus 100 nM 4-OHT. The luciferase activity obtained on transfection of pS2-Luc without exogenous ERα, ERΔ5 and HBx in the absence of E 2 was set as 1.

CC BY-NC

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2022-01-12 14:27:35

Nucleic Acids Res. 2006 Jun 6; 34(10):3095-3106

PMC1475750

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gkl389f4

Figure 4 HBx interacts with ERα in vitro and in vivo . ( A ) Interaction of HBx with ERα in vitro . A GST pull-down assay was performed using 35 S-labeled ERα, and GST or GST-HBx. The bound proteins were subjected to SDS–PAGE followed by autoradiography. ( B ) Interaction of HBx with ERα in vivo . ERα and FLAG-tagged HBx or empty vector were co-transfected into HepG2 cells. Cell lysates were immunoprecipitated (IP) by anti-FLAG M2 monoclonal antibody (Sigma), and the precipitates were then immunoblotted (IB) with anti-ERα polyclonal antibody (Santa Cruz Biotech). ( C ) Interaction of endogenous HBx with ERα in vivo . Liver tissue extracts from an HBV positive patient were immunoprecipitated with either anti-ERα polyclonal antibody or preimmune control serum (Santa Cruz Biotech). The precipitates were analyzed by immunoblot using anti-HBx (Chemicon). ( D ) Effect of ERΔ5 on the interaction between HBx and ERα. HepG2 cells were co-transfected with 2 μg ERα, 4 μg FLAG-tagged HBx and increasing amounts of ERΔ5 (2 and 4 μg). Cell lysates were immunoprecipitated by anti-FLAG monoclonal antibody, and the precipitates were detected with anti-ERα polyclonal antibody. ( E ) Co-localization of HBx and ERα in HepG2 cells. Cells were transfected with EGFP-tagged HBx and RFP-tagged ERα or empty vector (RFP) as indicated, and were treated with 10 nM E 2 for 24 h. The images were captured by confocal immunofluorescence microscopy. HBx localization is shown with EGFP (green) and ERα is seen with RFP (red). The nuclei were stained with DAPI (blue). Co-localization of HBx with ERα is shown in merged images. ( F ) Mapping of the ERα interaction region in HBx. A GST pull-down assay was performed using 35 S-labeled ERα and GST-HBx(1-72), GST-HBx(73-120), GST-HBx(121-154), GST-HBx(1-143), GST-HBx(52-154) and full-length GST-HBx(1–154) or GST. Schematic diagram of the HBx deletion constructs used is shown at the top, the binding of ERα to different regions of HBx is demonstrated in the middle, and SDS–PAGE analysis of the purified GST-fusion proteins is shown at the bottom. Asterisks indicate the positions of the expected purified GST or GST-fusion proteins. ( G ) Mapping of the HBx interaction region in ERα. A GST pull-down assay was performed using full-length GST-HBx(1–154) or GST, and 35 S-labeled full-length ERα (1–595), ERα (1–185), ERα (180–282), ERα (282–595), ERα (302–595) or ERΔ5. Schematic diagram of the ERα deletion constructs used is shown at the top.

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2022-01-12 14:27:35

Nucleic Acids Res. 2006 Jun 6; 34(10):3095-3106

PMC1475750

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gkl389f5

Figure 5 The HBx deletion mutant abolishes HBx-induced repression of ERα transcriptional activity. ( A ) Luciferase reporter assays with the HBx deletion mutants. HepG2 cells were co-transfected with 0.2 μg of ERE-LUC, 50 ng of the expression plasmid for ERα and 1.0 μg of the expression vector for FLAG-tagged HBx or HBx(Δ73–120), in the presence or absence of 10 nM E 2 . ( B ) Western blotting showing expression of FLAG-tagged HBx and HBx(Δ73–120). Cells were transfected as in (A). Cell extracts were prepared from E 2 -treated cells, and equivalent amounts of each extract were detected with anti-FLAG or anti-GAPDH antibody. ( C ) The HBx deletion mutant abolishes the HBx–ERα interaction. HepG2 cells were co-transfected with the expression plasmid for ERα and the expression vector for FLAG-tagged HBx or HBx(Δ73–120). Cell lysates were immunoprecipitated by anti-FLAG, and the precipitates were probed with anti-ERα.

CC BY-NC

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2022-01-12 14:27:35

Nucleic Acids Res. 2006 Jun 6; 34(10):3095-3106

PMC1475750

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gkl389f6

Figure 6 HBx forms a complex with ERα and HDAC1. ( A ) Association of HBx with HDAC1 in vitro . GST-HBx and GST were incubated with 35 S-labeled ERα, and a GST pull-down assay was then performed. ( B ) Association of HBx with ERα in vivo . HepG2 cells were transiently transfected with HA-tagged HDAC1 and FLAG-tagged HBx or control vector. Immunoprecipitation (IP) was performed using anti-FLAG monoclonal antibody; immunoblotting (IB) was performed with the indicated antibodies. ( C ) HBx interacts with both HDAC1 and ERα in vivo . HepG2 cells were co-transfected with ERα, HA-tagged HDAC1, and FLAG-tagged HBx or control vector. The cell extracts were immunoprecipitated with anti-FLAG monoclonal antibody followed by immunoblotting with the indicated antibodies. ( D ) HBx, ERα and HDAC1 forms a ternary complex. HepG2 cells were transfected as in (C). The cell extracts were immunoprecipitated with anti-FLAG antibody. Immune complexes were eluted with FLAG peptide and re-immunoprecipitated (re-IP) using anti-ERα polyclonal antibody and normal rabbit serum as a negative control. The resulting precipitates were resolved by SDS–PAGE followed by immunoblotting with the indicated antibodies.

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2022-01-12 14:27:35

Nucleic Acids Res. 2006 Jun 6; 34(10):3095-3106

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gkl389f7

Figure 7 Treatment of hepatoma cells with the specific HDAC inhibitor TSA causes a drastic relieving of HBx-induced repression of ERα transactivation. HepG2 cells co-transfected with 0.2 μg of the ERE-Luc reporter, 50 ng of the expression vector for ERα, 1.0 μg of the expression plasmid for HBx. Cells were then treated with control (0.1% ethanol) vehicle, 10 nM E 2 or 100 nM TSA as indicated. The luciferase activity obtained on transfection of ERE-Luc and ERα without exogenous HBx in the absence of E 2 and TSA was set as 1.

CC BY-NC

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2022-01-12 14:27:35

Nucleic Acids Res. 2006 Jun 6; 34(10):3095-3106

PMC1475751

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gkl392f1

Figure 1 Rapid fragmentation of nucleoid DNA into protein-linked HMW DNA fragments in E.coli treated with norfloxacin. Bacterial cells were treated with 0.1–100 μM norfloxacin for 30 min at 37°C, then were encapsulated in agarose plugs and subjected to lysis with SDS in the presence or absence of PK. Nucleoid integrity was then analyzed by PFGE as described in the Materials and Methods. [PK: protease K; Norf: norfloxacin].

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2022-01-12 14:27:35

Nucleic Acids Res. 2006 Jun 6; 34(10):3128-3138

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gkl392f2

Figure 2 Translation ( A ) and transcription ( B ) processes modulate HMW DNA fragmentation in E.coli treated with norfloxacin. Bacterial cells were pretreated for 30 min at 37°C with the indicated concentration of inhibitors of translation (kanamycin or chloramphenicol) or transcription (rifampicin), then for another 30 min with 10 μM norfloxacin in the continued presence of the inhibitor. The cells were then encapsulated and PFGE performed to examine the integrity of genomic DNA as described in the Materials and Methods. [Kan: kanamycin; Cm: chloramphenicol; Rif: rifampicin].

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2022-01-12 14:27:35

Nucleic Acids Res. 2006 Jun 6; 34(10):3128-3138

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Figure 3 Norfloxacin-induced excision of DNA loops is mediated by DNA topoisomerases. ( A ) Reversibility of norfloxacin-induced HMW nucleoid DNA fragmentation. Cells were treated for 30 min at 37°C with 10 μM norfloxacin, washed and encapsulated in agarose plugs. The plugs were then subjected to different reversal conditions in drug-free LB media for various times before analysis of fragmentation. ( B ) Coumermycin A1, a bacterial TOP2 catalytic inhibitor, inhibits norfloxacin-induced HMW nucleoid DNA fragmentation. Experiments were performed as described in Figure 2 , except that coumermycin A1 was used instead of translation or transcription inhibitors. [CouA1: coumermycin A1].

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2022-01-12 14:27:35

Nucleic Acids Res. 2006 Jun 6; 34(10):3128-3138

PMC1475751

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gkl392f4

Figure 4 Topo II is the target of coumermycin A1. ( A ) DNA gyrase is the target for coumermycin A1. ( B ) DNA gyrase, but not Topo IV, is mainly responsible for norfloxacin-induced HMW DNA fragmentation. Bacterial cells were treated with 50 μM coumermycin A1 for 30 min, then 10 μM norfloxacin was added and incubation continued for another 30 min at 30°C before PFGE was carried out to examine nucleoid integrity. [Cou A1: coumermycin A1; Norf: norfloxacin].

CC BY-NC

no

2022-01-12 14:27:35

Nucleic Acids Res. 2006 Jun 6; 34(10):3128-3138