EMBO/sd-character-level-ner huggingface dataset:
Figure 4Table reporting the qRT-PCR analyses of genes expression in control (shCTR) and antimiR-9 FaDu cells, as indicated. Data represent the mean (±SD) of three independent experiments performed in duplicate and unpaired t test was used to verify the statistical significance. On the left, WB analyses of KLF5 and Sp1 expression in control (shCTR) and antimiR-9 FaDu. Histone H3 expression was used as loading control. On the right, protein quantification analyses of KLF5 and SP1 expression normalized on H3 loading control. Data represent the mean (±SD) of three biological replicates and unpaired t test was used to verify the statistical significance. Schematic design of the KLF5 3'-UTR. To test the potential miR-9 binding on KLF5 3'-UTR, four vectors were generated: WT (Wild Type containing both the seed sites), mut A or mut B (mutated for one single binding site) and mut A+B (mutated in both seed site). The predicted seed sites for miR-9 binding sites are shown in red when present and in blue when mutated. Graph reporting the normalized Luciferase activity of wild type (WT) or mutated KLF5 3'-UTR described in C in control (shCTR) or antimiR-9 FaDu cells, as indicated. Data represent the mean (±SD) of three independent experiments performed in duplicate and unpaired t test was used to verify the statistical significance. qRT-PCR analyses of KLF5 (left) and Sp1 (right) expression in FaDu cells transfected with control or KLF5 vectors. Data represent the mean (±SD) of three independent experiments performed in duplicate and unpaired t test was used to verify the statistical significance. Upper panel shows WB analyses of KLF5 and Sp1 expression in FaDu cells. Actin expression was used as loading control. Lower graph shows the SP1 protein quantification normalized over actin expression. Data represents the mean (±SD) of three independent experiments and unpaired t test was used to verify the statistical significance. Graph reporting the normalized Luciferase activity of Sp1 promoter fragments in FaDu transfected with pcDNA control or KLF5 vectors, as indicated. Data represent the mean (±SD) of three independent experiments performed in duplicate and unpaired t test was used to verify the statistical significance. Chromatin immunoprecipitation (ChIP) assay performed on FaDu cells. Upper left panel shows WB analysis reporting KLF5 expression in the immunoprecipitation achieved using anti-KLF5 or control (IgG) antibodies used in the ChIP assay. Vinculin expression was used as loading control. Bottom left panel shows a typical image of amplified PCR fragments using ChIP DNA or genomic DNA, as indicated. Right graph reports the binding of KLF5 to the indicated region of Sp1 promoter expressed as signal relative to input in two independent immunoprecipitations (ChIP1 and ChIP2) using the KLF5 ab. IgG was used as negative control on the same chromatin. Primer pairs used to amplify the different regions of Sp1 promoter are depicted in the schema provided in Fig. 3I.
Pilot Screen for Known Regulators of Endosome-to-Golgi Retrieval Using Anti-CD8 Uptake Assay.Figure 1(A) Schematic of the anti-CD8 antibody retrieval assay as adapted for high-throughput screening. HeLa cells stably expressing a CD8-CIMPR reporter and the Golgi protein GFP-GOLPH3 were used. Anti-CD8 antibody (Ab) was bound at room temperature for 15 min and chased at 37°C for 30 min. Labeling and imaging details are in Supplemental Experimental Procedures.(B) Definition of the TGN retrieval ratio used in our studies and depiction of subcellular antibody localizations that give rise to high or low TGN retrieval ratios. (C) Scatterplots of the TGN retrieval ratios measured in replicate screens of 60 VPS gene homologs, 60 endocytosis genes, 43 SNARE protein genes, 41 dynein genes, 49 kinesin genes, and 43 myosin genes. Negative control measurements are in blue; SNX1 siRNA positive control measurements are in red. A linear fit to the data points is also shown. Marked data points indicate known endosome-to-Golgi retrieval pathway genes or genes that were selected for further validation.(D) Replicate TGN retrieval ratios for the validation screen of the pilot study, in which 80 individual ON TARGETplus siRNA sequences (four per gene) were assayed. Controls and linear fit are as in (C). (E) Representative images of the pilot validation screen for the negative control (no siRNA) and for five sets of siRNA-treated cells with reduced TGN retrieval ratio. Different phenotypes that give rise to reduced TGN retrieval ratios are discussed in the text. Scale bar, 100 μm (top two rows) and 38 μm (bottom row).
Figure 1mRNA expression of ANG in colonic tissues from normal controls (n = 25) and UC (n = 27) or CD (n = 46) patients. Representative western blotting of ANG in colonic tissues from normal controls and UC or CD patients. Representative images showing ANG immunohistochemical (IHC) staining in normal control and UC or CD sample. Corresponding statistical analysis of ANG expression score in normal controls (n = 10) and UC (n = 10) or CD (n = 15) patients. Representative images showing hematoxylin-eosin (HE) staining and ANG IHC staining in mild and severe IBD samples. Statistical analyses of ANG expression scores in mild (n = 18) and severe (n = 19) UC (F) or mild (n = 20) and severe (n = 20) CD (G) patients.
STING-mediated IFN induction and attenuation of HSV1 replication and pathogenesis require EGFR.Figure 2(A) MEF cells were pre-treated with gefitinib or DMSO for 1 hour, and then infected with HSV1 at m.o.i. of 1. 4 hours post infection (h.p.i.), IFNβ mRNA induction was monitored by qRT-PCR (n=3). Uninfected cells were used as negative control (U). (B) DNA was extracted from gefitinib-treated or untreated infected MEFs 0 and 16 h.p.i. and viral DNA was measured by qPCR (n=3). (C) WT and EGFR KO HeLa cells were infected with HSV1, RNA was extracted at 4 h.p.i. and IFNα mRNA induction was measured (n=3). (D) Survival of uninfected mice treated with gefitinib and HSV1-infected mice treated or untreated with gefitinib. (E) Survival of WT and EGFRfl+/-LysMCre+/+ mice infected with HSV1. WT and EGFRfl+/-LysMCre+/+ mice infected with HSV1. HSV1 titers in the brains of the live mice at day 9 were determined.
– Division of labor model of the regulation of cell proliferation and DNA damage response in Arabidopsis.Figure 8DNA damage, e.g. induced by chemical mutagens, is followed by the activation of the checkpoint kinase ATM (Ataxia Telangiectasia Mutated) that activates the transcription factor SOG1 (Suppressor of Gamma Response 1). On the one hand, SOG1 represses, directly or indirectly, the expression CDKB2 and possible other CDKs as the major driving force of mitosis to allow a cell time for DNA repair. On the other hand, SOG1 directly binds to the promoter of CYCB1;1 and activates its expression. CYCB1;1 builds an active complex together with CDKB1. This complex phosphorylates the DNA binding protein RAD51 (Radiation Sensitive 1) that gets recruited to the DNA damage site. This cascade is required for HR in S and G2 phases of the cell cycle in meristematic cells. To trigger this response, the action of all four B1-type cyclins is necessary, possibly by providing a threshold of mitotic CDK activity that then gets amplified through SOG1-dependent stimulation of CYCB1;1 expression.
Figure 5Stomatal aperture 1 h (A) or 4 h (B) after spraying with Pst DC3000 or mock treatment. (C) Microscope images of stomata at 1 and 4 hpi with Pst DC3000 (Mock at 4 h). (D), Stomatal aperture 1 h after treatment with COR, ABA, or COR plus ABA, compared to mock. In (A), (B), and (D), data are means ± SD of 12 to16 measurements; in each measurement the rosette leaves from at least 6 - 8 plants were used). Asterisks indicate a significant difference from mock treatment (p < 0.0001; Student´s t-test).
Formin inhibition abolishes nuclear import A Confocal planes of nuclei of cells treated with DMSO (Ctl) or SMIFH2 (50μΜ) for 4h, stained with mAb414 and DAPI (DNA).Figure 2
a) Mitochondrial tubules in HeLa cells (top, TMRE stained) exceeded the dimensions of that for autophagosomes (labelled by EGFP-LC3B) under either normal (middle, same field as TMRE image) or starvation conditions (right).f1(a) Mitochondrial tubules in HeLa cells (top, TMRE stained) exceeded the dimensions of that for autophagosomes (labelled by EGFP-LC3B) under either normal (middle, same field as TMRE image) or starvation conditions (right). (b) Diagram of light-induced mitophagy. Single mitochondrial tubules containing KR-dMito, when illuminated by 559 nm light, are selectively impaired, leading to surface recruitment of parkin and subsequent autophagic turnover. (c) Illuminating a selective region (white dotted circle, top) within a KR-dMito/EBFP2-parkin-expressing HeLa cell led to the immediate loss of KR fluorescence (top two panels). Third to fifth row: magnified view of the photo-impaired region, EBFP2-parkin translocated onto impaired mitochondrial tubules synchronously (38-60 min after 559 illumination, white arrows). Scale bars (all panels), 10 μm.
Proposed models on Chk1-dependent control of DNA replication and its effect on cell proliferation.Figure 9Model in which excess CDC45 creates replication barriers in Chk1-deficient cells. These barriers recruit Polη, which remains inactive due to CDK-mediated phosphorylation. Model in which reduced origin firing and Polη-independent increased fork elongation promote proliferation in Chk1-deficient cells.
in the cytosol (A) Immunoblot analysis of proteins precipitated with anti-EphA2 or IgG (control) were from whole-cell lysates of AECs from wild-type mice stimulated with reovirus (MOI=5) for 3 h.Figure 5(A) Immunoblot analysis of proteins precipitated with anti-EphA2 or IgG (control) were from whole-cell lysates of AECs from wild-type mice stimulated with reovirus (MOI=5) for 3 h. Input, 10% of the AECs lysate. Data information: The position of protein markers (shown in kDa) is indicated on the right-hand side. Data are representative of three independent biological replicates. (B) Full-length EphA2 and serial truncations of EphA2 with deletion of various domains. (C) The indicated plasmids of HA-tagged full-length EphA2 and EphA2 truncation mutants were respectively transfected into HEK293T cells along with Myc-tagged NLRP3, and then cell lysates were immunoprecipitated with anti-Myc antibody, followed by western blot analysis with anti-HA antibody and anti-Myc Antibody. Data information: The position of protein markers (shown in kDa) is indicated on the right-hand side. Data are representative of three independent biological replicates. (D) The indicated plasmids of HA-tagged full-length NLRP3 and NLRP3 truncation mutants were respectively transfected into HEK293T cells along with Myc-tagged EphA2, and then cell lysates were immunoprecipitated with anti-Myc antibody, followed by western blot analysis with anti-HA antibody and anti-Myc antibody. Data information: The position of protein markers (shown in kDa) is indicated on the right-hand side. Data are representative of three independent biological replicates. (E) Colocalization of endogenous EphA2 and NLRP3 in AEC cells. AEC cells were unstimulated or stimulated with reovirus for 2h before confocal microscopy analysis. EphA2 was stained with rabbit anti-EphA2 (1:500), followed by Alexa Fluor 594 goat anti-rabbit secondary antibody (red), while NLRP3 was stained with mouse anti-NLRP3 (1:500), followed by Alexa Fluor 488 goat anti-mouse secondary antibody (green). DAPI (4′,6-diamidino-2-phenylindole) served as the nuclei marker (blue). Scale bars represent 20μm. The Pearson's correlation coefficient and the Mander's overlap coefficient in the upper red box are 0.5, and 0.95 respectively, and the lower red box 0.78, and 0.94 respectively.
SSADH‐deficient mice have increased numbers of mitochondria and aberrant antioxidant levels that can be normalized by rapamycinElectron microscopy images of mitochondria from WT (n = 44) and SSADH-deficient mice (Aldh5a1-/-) (n = 80) were calculated for area size.Figure 8A Electron microscopy images of mitochondria from WT (n = 44) and SSADH‐deficient mice (Aldh5a1−/−) (n = 80) were calculated for area size. B Electron microscopy images showing typical sizes of WT and Aldh5a1−/− mice liver mitochondria. Bar, 0.5 μm. C Quantification of mitochondrial numbers from electron microscopy images of liver from WT (n = 31) and Aldh5a1−/− mice treated with vehicle (n = 39) or rapamycin (n = 34) (5 mg/kg body weight per day) via intraperitoneal injections for 3 successive days starting at day 7 of life. D Quantification of mitochondrial numbers from electron microscopy images of brain from WT (n = 23) and Aldh5a1−/− mice treated with vehicle (n = 30) or rapamycin (n = 41) (5 mg/kg body weight per day) via intraperitoneal injections for 3 successive days starting at day 7 of life. E Aldh5a1−/− mice were treated with vehicle or rapamycin (10 mg/kg body weight per day) via intraperitoneal injections for 10 successive days starting at day 10 of life. WT mice served as non‐disease controls (set to 1). After sacrifice, liver homogenates were used to measure SOD enzyme activity using a colorimetric SOD activity assay. F Mitochondrial SOD2 protein levels were quantified from liver microsections using immunofluorescence microscopy and automated image analysis (WT set to 1). G Immunofluorescence images showing typical nuclear staining (DAPI, blue) and SOD2 staining (red) from WT, Aldh5a1−/− mice treated with vehicle and Aldh5a1−/− mice treated with rapamycin. Bar, 10 μm.
Figure 7A-C, Micrographs showing GFP (green: electroporated cells (A-C)) and Ncl (grey: nucleoli (A', B' and C')) immunostaining of E15 cerebral cortex 2 days after IUE with plasmids indicated. Position of magnifications shown on the right side are depicted by a white square. V= ventricle; VZ: ventricular zone. Yellow dotted shapes show examples of nucleoli in electroporated cells from the VZ and the non-VZ. D-F Violin plots depicting the nuclear size (D) nucleoli number (E) and nucleoli size (F) from cells electroporated as in A-C with the constructs and the areas indicated on the x-axis. Note that in Trnp1, but not ∆1-16Trnp1, overexpression is sufficient to significantly increase nucleolar size in the non-VZ (where most cells lack endogenous Trnp1). N=10 cells per region from 6 different control and Trnp1-IRES-GFP electroporated embryos and N=15 per region from 3-4 different ∆1-16Trnp1-IRES-GFP electroporated embryos from at least 4 litters. G-H, Micrographs showing GFP (green: electroporated cells), Pax6 (magenta for NSCs) and Ncl (grey for nucleoli) immunostainings in sections of E15 cerebral cortex 2 days after IUE with plasmids indicated. Yellow dotted shapes show examples of nucleoli in electroporated and yellow non dotted shapes show examples of nucleoli in non-electroporated cells from the VZ and the non-VZ. I-K Violin plots depicting the nuclear size (D) nucleoli number (E) and nucleoli size (F) from cells electroporated as in G-H with the constructs and the areas indicated on the x-axis. Note that Trnp1 knockdown significantly decreases nucleolar size in the VZ (where most cells express endogenous Trnp1). N=10 cells per region from 7 embryos per condition from at least 4 litters.
a) vBcl-2 binding proteins.figf1(a) vBcl-2 binding proteins. After transfection, 293T cells were used for GST-pulldown and the purified proteins were detected by silver staining. (b) Schematic representation of vBcl-2, Beclin1, PI(3) KC3 and UVRAG protein structure. BD, Bcl-2 binding domain; Acc, accessory domain; BH1, Bcl-2 homologue 1. (c) UVRAG binds Beclin1 through its CCD. After transfection with Beclin1-V5, together with full-length or fragments of Flag-UVRAG, 293T whole cell lysates (WCLs) were used for immunoprecipitation (IP) with anti-Flag followed by immunoblotting (IB) with anti-V5. (d) Interactions between endogenous UVRAG and Beclin1 and between endogenous UVRAG and PI(3) KC3. 293T cell lysates were used for immunoprecipitation with preimmune sera or anti-Beclin1, followed by immunoblotting with anti-UVRAG or anti-Beclin1, or for immunoprecipitation with preimmune sera or anti-UVRAG, followed by immunoblotting with anti-PI(3) KC3. (e) UVRAGCCD is required for Beclin1 interaction. After transfection with Beclin1-V5 (lanes 2 and 4) together with Flag-UVRAGΔCCD (lanes 1 and 2) or Flag-UVRAGΔC2 (lanes 3 and 4), WCLs were used for immunoprecipitation with anti-Flag followed by immunoblotting with anti-V5. (f) Beclin1 binds UVRAG through its CCD. After transfection with Flag-UVRAG together with full-length or fragments of Beclin1-V5, WCLs were used for immunoprecipitation with anti-V5 followed by immunoblotting with anti-Flag. (g) An increase in UVRAG stability induced by Beclin1. After transfection with Flag-UVRAG and empty vector or Flag-UVRAG and Beclin1-V5, WCLs were treated with cycloheximide (CHX, 20 μg ml−1) for the indicated time, followed by immunoblotting with anti-Flag. (h) UVRAG directly interacts with Beclin1. MBP-UVRAGCCD (lane 1), His6-Beclin1CCD (lane 2) or mixtures of both proteins in a concentration dependent manner (lanes 3-8), were subjected to native gel electrophoresis. The numbers indicate the concentration (nmol) of protein. Arrows indicate the purified MBP-UVRAGCCD, His6-Beclin1CCD and complex between these two. (i) Beclin1CCD and UVRAGCCD associate into a α-helical heterodimer. After removing the N-terminal His6 and MBP tags, the Beclin1CCD and the preformed Beclin1CCD-UVRAGCCD complex were subjected to circular dichroism spectroscopic analysis. The raw data for this experiment are presented in the Supplementary Information, Fig. S5.
a) Nuclear localization of HLH-30 was quantified in day 1 adult animals expressing HLH-30::GFP.f2(a) Nuclear localization of HLH-30 was quantified in day 1 adult animals expressing HLH-30::GFP. Animals were fed bacteria expressing control or tor dsRNA from hatching and raised at 20 °C (three biological replicates, ~50 animals each, mean±s.d., **P0.01, Student's t-test). b,c) Expression of putative autophagy-related and lysosomal target genes was measured by quantitative PCR in day 1 (b) WT (N2) and (c) hlh-30(tm1978) animals fed bacteria expressing control or tor dsRNA from hatching (20 °C). Data are mean±s.d. of biological triplicates. *P0.05, **P0.01; Student's t-test. csta* is a cathepsin A orthologue (cosmid C08H9.1 (ref. 30)). (d) Lifespan analysis of WT animals and hlh-30(tm1978) mutants fed bacteria expressing control or tor dsRNA from day 1 of adulthood was carried out at 20 °C. See Supplementary Table S3 for details of lifespan analyses and replicate experiments.
Figure 4A. Kinase Enrichment Analysis showing enrichment of TAK1 (encoded by MAP3K7) in injured AECs (p-adj < 0.05, hypergeometric test with Benjamini-Hochberg Correction). B. Nuclear fractionation followed by simple western analysis (Peggy Sue) of AECs exposed to 72 h of TGFβ1 in the co-culture system shows an increase in phosphorylated TAK1 and a parallel increase in ph-EZH2. Quantifications (lower panels) show mean + s.d., n = 3 biological replicates (ns = non-significant, ***p < 0.001, Kruskal-Wallis/Dunn's). C. Simple western analysis (Peggy Sue) of EZH2 immunoprecipitates shows increased co-precipitation of ph-EZH2 (T311) and ph-TAK1 in injured AECs. Unspecific IgG was used as negative control. A representative from three experiments is shown. D. Simple western analysis (Peggy Sue) shows a TAK1-dependent enrichment of ph-EZH2 levels in injured AECs. Note: TAK1 inhibitor (5-OZ) attenuates increased ph-EZH2 levels in injured AECs. Quantifications (right panels) show mean + s.d., n = 5 biological replicates (ns = non-significant, **p = 0.0026, ***p < 0.001, ANOVA/Tukey's). E. ChIP-qPCR shows diminished POL2 occupancy on profibrotic genes in injured AECs subjected to 5-OZ (mean + s.d., n = 3 biological replicates). Unspecific IgG was used as negative control.
is downregulated in CRC samples.Figure 7A Twenty pairs of fresh frozen CRC tissues and corresponding normal tissues were lysed and total RNAs were extracted and subjected to RT-qPCR. (N, Normal tissue; T, Tumor tissue) **p<0.01 (non-parametric Mann-Whitney t-test) B Immunohistochemical staining of NAT10 in CRC tissues and normal colon tissues. NAT10-specific immuno-complex was detected with DAB as shown as brown color. Quantification of NAT10-positive or NAT10-negative CRC cases is shown in the lower table. C Cell extracts were prepared from different human colon epithelial cell lines as indicated at the top. Proteins from the extracts were subjected to western blot for the evaluation of NAT10 and p53. D CRISPR-Cas9 system mediated NAT10 gene editing. The genome targeting for knockdown NAT10 was performed as described in Experimental Procedures. The HCT116 NAT10 knockdown cell lines were harvested and western blot was performed on the cell lysates for the indicated proteins. E HCT116 Ctrl or c2-4 cells with NAT10 disruption were treated with actinomycin D and harvested at the indicated time points. Cell cycle profiles were determined by FACS. Results from three independent experiments are shown as mean ± SEM. (two-tailed Student's t test) F HCT116 Ctrl cells or c2-4 cells with NAT10 disruption were treated with actinomycin D Cells were harvested at the indicated time points and lysed. The total proteins were analyzed by western blot using the indicated antibodies. G HCT116 Ctrl or c2-4 cells were irradiated with 80 J/m2 UV. Forty-eight hours later, apoptotic cells were determined. Results from three independent experiments are shown as mean ± SEM. (two-tailed Student's t test) H Colony formation assay were performed with HCT116 cells (Ctrl, c1-1 and c2-4). The right panel shows the quantification of formed colonies. (two-tailed Student's t test) I HCT116 c1-1 and c2-4 cell lines were plated and cell growth curves were determined as described in Fig 6F. Error bars represent the SEM of the triplicate experiments. J The working model of p53 regulation by NAT10.