EMBO/sd-character-level-ner huggingface dataset:
Figure 1A. HeLa, H1299 and A549 cells were infected with lentiviruses expressing control shRNA or lncRNA-MIF shRNA. Forty-eight hours after infection, cell lysates were subjected to Western blot analysis with anti-c-Myc antibody. B. Schematic illustration of lncRNA-MIF genomic localization and three putative c-Myc binding sites. C. Total RNA from the indicated cell lines was subjected to Northern blot analysis to determine the molecular size of lncRNA-MIF. Actin was also included as a positive control. D. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF, control shRNA, or lncRNA-MIF shRNA as indicated. Forty-eight hours after infection, cell lysates were analyzed by Western blotting with anti-c-Myc and anti-GAPDH antibodies. E. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF, control shRNA, or lncRNA-MIF shRNA as indicated. Forty-eight hours after infection, total RNA was extracted from these cells and subjected to real-time RT-PCR analysis. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test). F. HeLa cells expressing either control RNA or lncRNA-MIF were treated with or without MG132 (20 μM) for 6 h. Cell lysates were analyzed by Western blot with the indicated antibodies. G. HeLa cells expressing either control shRNA or lncRNA-MIF shRNA were treated with MG132 for 6 h. Cell lysates were analyzed by Western blotting with the indicated antibodies. H. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF or lncRNA-MIF-AS as indicated. Forty-eight hours after infection, cells were treated with cycloheximide (CHX, 50 μg/ml) for the indicated periods of time. Cell lysates were then analyzed by Western blotting to examine the c-Myc protein half-life. Protein band intensity were analyzed by ImageJ.I. Band intensity of c-Myc in Fig 1H was analyzed by ImageJ. J. HeLa cells were infected with lentiviruses expressing control shRNA, lncRNA-MIF shRNA-1, -2 or -3 as indicated. Forty-eight hours after infection, cells were treated with CHX for the indicated periods of time. Cell lysates were then analyzed by Western blotting to examine the half-life of c-Myc protein.K. Band intensity of c-Myc in Fig 1J was analyzed by ImageJ.
Figure 4A Macrophages (cytoplasm, red) near the germband in Stage 11/12 embryos show partial colocalization of the HA-antibody labeling Porthos (green) with the nucleus (DAPI, blue). Data information: Movies in each analysis set are from independent embryos. Scale bars: 50µm B Stills starting at Stage 11 from two-photon movies of control embryos and those expressing porthos (pths)-RNAi in macrophages (nuclei, red), migrating from the head mesoderm towards and into the germband at the indicated time points. White dotted line: germband edge. Data information: Movies in each analysis set are from independent embryos. Scale bars: 30µm C-H Quantification of macrophage migration parameters from movies as in (B). Macrophage migration speed (C) in the head or (D) between the yolk sac and the germband edge. (C) control n=4 movies, pths RNAi n=6; control n=507 tracks, pths RNAi n=859; p=0.56. (D) control n=5 movies, pths RNAi n=5; control n=40 tracks, pths RNAi n=51; p=0.45. (E) The time required for the first macrophage nucleus to enter into the extended germband: control=23 min, CG9005PBG=38 min, p<0.0001. The migration speed of the (F) 1st, (G) 2nd, or (H) 3rd-5th macrophages along the first 25-30µm into the germband between the mesoderm and ectoderm: control=21.5 min, n=6, pths RNAi=36.2 min, n=4, p<0.0001. Data information: Mean±SEM, ns=p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Unpaired t-test I Confocal images of early Stage 12 embryos from control, atosPBG, and atosPBG expressing atos::FLAG::HA (mac>atos) or pths::FLAG::HA (mac>pths) in macrophages (red). Embryo detected by phalloidin staining (green). White dotted line: germband edge.. J Quantification of macrophages in the germband of an atosPBG mutant rescued by expressing pths::FLAG::HA in macrophages. For control (n=15 embryos) vs atosPBG mutant (n=22) and mac>atos rescue (n=27) both p<0.0001, vs mac>pths rescue (n=30) p=0.0007. Data information: Movies in each analysis set are from independent embryos. Mean±SEM, ns=p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. one-way ANOVA with Tukey Scale bars: 50µm
through ubiquitin chains (A) RAW264.Figure 7(A) RAW264.7 cells were pretreated with or without E64d/PepA or MG132 for 4 hours, and then stimulated with or without Mtb for 12 hours as indicated. Cell lysates were collected and immunoprecipitated using an anti-RIPK3 antibody, and the ubiquitination of endogenous RIPK3 was detected using an anti-Ub antibody. Representative blots from n = 3 biological replicates are shown. (B, C) Socs6+/+ BMDMs were transfected with miR-342-3p mimic or Socs6 siRNA (B), and Socs6-/- BMDMs were transfected with plasmid expressing Myc-SOCS6 (C) for 24 hours as indicated. Transfected cells were then stimulated with Mtb for another 12 hours, cell lysates were collected for immunoprecipitation, and ubiquitination of endogenous RIPK3 was detected by immunoblot. Representative blots from n = 3 biological replicates are shown. (D) RAW264.7 cells were transfected with A20 siRNA, cell lysates were collected for immunoprecipitation and immunoblot. Representative blots from n = 3 biological replicates are shown. (E-G) RAW264.7 cells were transfected with plasmids expressing A20 and RIPK3 (E), or A20 and RIPK3 mutants (G) as indicated, cell lysates were collected for immunoprecipitation and immunoblot. Myc-A20 or Flag-RIPK3 purified from transfected HEK-293T cells was incubated with ATP, E1, E2 and ubiquitin as indicated. The in vitro ubiquitination of RIPK3 was analyzed by immunoblot using an anti-Ub antibody (F). Representative blots from n = 3 biological replicates are shown.
Identification of Oct1/2 binding sites in the promoters of the Btla and Bcl6 genes.Figure 7(A) In silico search for potential Oct1/2 binding sites within the first 2000 bp upstream of the transcriptional start site of the Btla and Bcl6 genes (marked in red and green, respectively). Nucleotides that differ from the consensus octamer sequence ATGCAAAT are marked in blue. (B and C) Inducible complex formation on the potential octamer sites within the Btla and Bcl6 promoters. Purified murine CD4+ T cells were left untreated or induced with PMA and Ionomycin (P/I) for 18h. Whole cell extracts were analyzed by EMSA using labeled, double-stranded oligonucleotides containing either one of the potential octamer motif of the Btla or Bcl6 promoters as depicted in (A) or the consensus octamer site that served as an internal control. (D) Competition analysis for complex formation on potential Oct binding sites in the Btla and Bcl6 promoters. EMSA analysis of whole cell extracts from purified CD4+ T cells stimulated for 18 h with P/I. Radioactively labeled, double-stranded oligonucleotides containing one of the potential octamer binding sites of the Btla and Bcl6 promoters were added to the reaction mixture together with a 10x or 100x molar excess of non-labeled oligonucleotides comprising either the consensus octamer motif or the consensus NF-kB motif.
Figure 5A. HEK 293T cells were transfected with plasmids as indicated for 24 hr and treated with DMSO or 200 nM ISRIB for 2 hr followed by analysis of the protein level of CHOP, p-eIF2α, total eIF2α, and Endou-Flag using Western blot. The α-tubulin served as an internal control. Protein levels relative to each internal control are presented in each lane. B. HEK 293T cells were transfected with plasmids as indicated for 24 hr and treated with DMSO, thapsigargin (TH), ISRIB, or TH plus ISRIB for 2 hr followed by analysis of the protein level of CHOP, p-eIF2α, total eIF2α, Endou-Flag, and its variants using Western blot. The α-tubulin served as an internal control. Protein levels relative to each internal control were presented at each lane. C. MEF wild-type (WT) and its eIF2αS51A mutant (S51A) cells were treated with 1 μg of TH for 4 hr followed by Western blot analysis of p-eIF2α, total eIF2α, and ENDOU. GAPDH served as an internal control. Protein levels relative to each internal control are presented in each lane. D, E. MEF WT and mutant S51A cells were transfected with plasmids as indicated for 24 hr and treated with either DMSO or TH for 4 hr followed by analysis of the protein levels of CHOP, p-eIF2α, total eIF2α, GADD34, and Endouc-Flag using Western blot. GAPDH served as an internal control. Protein levels relative to each internal control were presented at each lane. F. HEK 293T cells were transfected with negative-control siRNA and ENDOU-siRNA for 48 hr. After treatment, protein lysates were prepared and subjected to Western blot analysis for ENDOU-1 and CHOP detection using antibodies. The α-tubulin served as an internal control. Protein levels relative to each internal control were presented in each lane.
Figure 3A. Immunoblot analysis of U2OS/GFP-FAM111A WT cells treated with DOX for the indicated times. B. Representative images of U2OS/GFP-FAM111A cell lines that were treated or not with DOX, fixed and stained with γ-H2AX antibody and DAPI. C. Cells in (B) were subjected to QIBC analysis of γ-H2AX signal intensity (red bars, mean (A.U., arbitrary units); n>2000 cells per condition). D. Immunoblot analysis of U2OS/GFP-FAM111A WT cells treated or not with DOX and the pan-Caspase inhibitor Z-VAD-FMK as indicated. Transcriptional activity in U2OS/GFP-FAM111A cells treated or not with DOX for the indicated times were analyzed by QIBC (red bars, mean; n>2000 cells per condition). Transcriptional activity in U2OS/GFP-FAM111A cells treated or not with DOX pulse-labeled with EU were analyzed by QIBC (red bars, mean; n>2000 cells per condition). Transcriptional activity in U2OS/GFP-FAM111A cells treated or not with DOX pulse-labeled with EU and stained with DAPI were analyzed by QIBC (red bars, mean; n>2000 cells per condition). H. U2OS/GFP-FAM111A cell lines treated or not with DOX were pre-extracted, fixed and stained with RPB1 antibody, and analyzed by QIBC (n>2000 cells per condition). I. Quantification of data in (H) (red bars, mean). U2OS/GFP-FAM111A cell lines treated or not with DOX were pre-extracted, fixed and stained with RPB1 antibody, J. Representative images from the experiment in (H). Scale bars, 10 μm.
Figure 8Schematic diagram depicting how mTORC1-eIF4F controls mESC self-renewal, and the mTORC1-independent but MEK/ERK-dependent role of eIF4F in SL to regulate pluripotency.
a) PC12 cells stably expressing A53T mutant α-synuclein were induced for 24 h, after which expression was switched off by removing doxycycline from the medium for the next 24 h.figf1(a) PC12 cells stably expressing A53T mutant α-synuclein were induced for 24 h, after which expression was switched off by removing doxycycline from the medium for the next 24 h. Transgene product clearance can be inferred by assessing levels at various times after switching off expression after an initial induction period, as expression levels decay when synthesis is reduced or stopped. Cells were treated with EHNA or AMP-PNP or left untreated during the switch-off period. Blots were probed with antibody to HA to detect the transgene and antibody to actin as a loading control. (b) PC12 cells expressing Q74-GFP were induced for 15 h and then treated with inhibitors for 48 h after switch-off. Band intensity quantification (three independent experiments) is shown. (c) Immunofluorescence images of cells expressing Q74-GFP after 48 h of switch-off after treatment with (EHNA) or without (Control) EHNA. (d) Quantification of aggregates and cell death in COS-7 cells transiently transfected with Q74-GFP either with (EHNA) or without (Control) EHNA (added during the last 33 h before fixation). (e) Western-blot analysis of HeLa cells stably expressing GFP-UbG76V treated with lactacystin (Lac), EHNA or AMP-PNP or left untreated (Control) using antibody to GFP. ***P 0.0001; **P 0.001.
Fig.Figure 1Defects in midbrain-type dopamine (mDA) neuron-specific developmental factor expressions in the ventral midbrain (VM) of Lin28a/b cKO embryos. Embryonic VM sections from 3 WT and cKO animals each were immunoflorescent stained for the midbrain-specific markers in parallel and mean fluoresecent intensities (MFI) were measured. n = 20 cells for each group. *** p<0.0001, t-test. Reduced mDA neurons in the embryonic VM (E12.5) and adult SN at 2-7 months (young age) and at 14-18 months (old age) of the Lin28a/b cKO mice. *p<0.05, **p<0.005, n=4 each wild-type (WT) and cKO embryos, and 9 (WT), 8 (cKO) in adult mice. Enhanced DA transporter (DAT)+ and TH+ fiber intensities of Lin28a/b cKO adult mice (age: 4-7 months). Striatal sections of WT and cKO mice (3 animals for each group) were subjected to immunofluorescent staining against DAT and TH in parallel, and MFI was measured from 101 microscopic fields of dorsolateral striatum. DA uptakes assessed by nanoScanPET/MRI analysis using [18F]FP-CIT. n=9 (WT), 8 (cKO). *p<0.05, ***p<0.001. Enhanced susceptibility of Lin28a/b cKO mice to the Parkinsonian toxin MPTP. WT and cKO mice were injected with MPTP (20mg/kg, i.p) for 5 days and behaviors of the animals were assessed for 1 month after the MPTP injection Enhanced susceptibility of Lin28a/b cKO mice to the Parkinsonian toxin MPTP. The mouse brains at 1 month after initial MPTP exposure were subjected to immunohistochemical analyses for TH+ mDA neuron counts at SN (L) and DAT MFI at striatum (M).
Figure 4(A) The degradation of 3HA-Pgc1 was analyzed in cells with the indicated genotype as in Fig. 1A. The graph shows the average of three independent experiments; error bars represent the standard deviation. (B) The degradation of the Doa10 substrates 3HA-Pgc1 and 3HA-Vma12-Ndc10C' was analyzed in cells of the indicated genotypes treated with oleic acid. 3HA-Pgc1 and 3HA-Vma12-Ndc10C' were detected with anti-HA antibodies. Dpm1 was used as a loading control and detected with anti-Dpm1 antibodies. The graph shows the average of three independent experiments; error bars represent the standard deviation.
Figure 7Under normal cell growth conditions, the pre-ribosomal complex formed by RPL5, RPL11 and 5S rRNA is incorporated into nascent ribosomes (Left panel). When nucleotide levels become limiting for 47S rRNA synthesis, ribosome biogenesis is impaired, which converts the RPL5/RPL11/5S rRNA complex into the IRBC complex, which is able to bind and inhibit MDM2, releasing p53, leading to p21 induction and G1 arrest (Middle panel). However, a more severe drop in nucleotide levels leads to the degradation of p21 by the proteasome, impairing IRBC-dependent G1 arrest and allowing S phase entry despite reduced nucleotide pools. Cells are therefore exposed to replicative stress, sensed by the ATR-Chk1 pathway responsible for p53 S15 phosphorylation. Chronic replicative stress leads to DNA-damage and eventually genomic instability, key events in tumor progression (Right panel).
Figure 2A, B: correlations between S1P and HDL-C (A), and albumin (B). Data information: Scatter plots and fitted regression line are shown in each figure. Each S1P measurement was run in triplicate, and performed at least twice in independent assays. Pearson correlation was performed for statistical analysis. Exact p values or ****p < 0.0001 are reported in the figure. C, D: correlations between apoM and HDL-C (C) and albumin (D). Data information: Scatter plots and fitted regression line are shown in each figure. Each apoM measurement was run in triplicate, and performed at least twice in independent assays. Pearson correlation was performed for statistical analysis. Exact p values or ****p < 0.0001 are reported in the figure.
Domain analysis of the CbpC-p62 interaction.Figure 5A Schematic representation of the CbpCT4 variants used in (B), (E), (H) and (I). B Lysates from 293T cells transiently expressing p62-3Myc and GFP-CbpCT4 variants were immunoprecipitated using a GST-GFP-Nanobody fusion protein. Bound proteins were analyzed by immunoblotting. C Diagram of the ∆DD mutation in dp5 loop domain. D Structures of the dp5 and dp6 domains in CbpFR6. The side chains of D167-D168 are shown. E Lysates from 293T cells transiently expressing p62-3Myc and GFP-CbpCT4 variants were immunoprecipitated using the GST-GFP-Nanobody protein. Bound proteins were analyzed by immunoblotting. F Diagram of the p62 variants used in (G). G Lysates of 293T cells transiently expressing GFP-CbpCT4 and p62-3Myc variants were immunoprecipitated with the GST-GFP-Nanobody protein. Bound proteins were analyzed by immunoblotting using the indicated antibodies. H A549 cells infected with the indicated S. pneumoniae strains for 2 h were fixed and stained with DAPI and an anti-Atg14 antibody, and the percentages of perinuclear-localizing Atg14 containing cells were quantified. I Representative epifluorescence images in (H) are shown. Scale bars, 10 µm.
a) Knockdown of RARα in NIH3T3 mouse fibroblasts was conducted using two different shRNAs, sh1 and sh2, compared to control (Ctr).figf1(a) Knockdown of RARα in NIH3T3 mouse fibroblasts was conducted using two different shRNAs, sh1 and sh2, compared to control (Ctr). Left, representative immunoblot. Actin is shown as loading control and full-length blots are shown in Supplementary Figure 21. Right, amounts of RARα in control and knockdown cells determined by densitometric quantification of immunoblots represented by the one shown on the left. Values are normalized for actin and expressed as multiples of control (None) values; n = 3. (b) Rates of degradation of long-lived proteins in control and RARα(-) cells maintained in the presence or absence of serum for 12 h. Values are expressed as percentage of proteolysis; n = 3. (c,d) Percentage of degradation due to lysosomes (c) and macroautophagy (d) in cells assayed as in b but treated with inhibitors of lysosomal proteolysis (c) or with 3-methyladenine (3MA) to block macroautophagy (d). Values are expressed as percentage of total protein degradation sensitive to the lysosomal inhibitors; n = 3. In all panels, all values are mean ± s.e.m., and differences with control are significant for *P 0.05.
Figure 5(A) Super resolution microscopy (SIM) imaging of Flag-tagged proteins, cyclophilin A (CypA-Flag), EB1-Flag, or Flag-CLIP170 bound to in vitro assembled CA-NC complexes, stained with anti-p24 AG3.0 (Red), anti-Flag or anti-CLIP170 (Green). Scale bar, 2 μm. Representative images from different fields of view are shown. (B) WB analysis confirming protein expression in samples from A and C. Molecular weight markers (in kDa) are shown to the right of WBs. (C) Quantification (n=3) of the association of each protein with 97 to 137 CA-NC complexes, using untransfected lysates (Control) and EB1-Flag as controls for non-specific background staining. (D) Lysates from untransfected, CypA-Flag- or Flag-CLIP170-transfected cells were subjected to CA-NC binding assays and WB analysis. (E) Levels of CypA or CLIP170 bound to WT or R18L CA-NC assemblies in samples from D were quantified using Fiji (n = 4 experimental replicates; data is presented as a ratio of CypA or CLIP170 in bound versus input for each CA-NC assembly, normalized to WT CA-NC arbitrarily set to 1). Statistical significance was determined by t test (mean ±SEM). *** = p<0.001.
Endosome types involved in FP+.Figure 6Endosome types involved in FP+. The FP+ were classified according to MVE and VE involved. The actual numbers of FP+ analyzed are given above the bars.
Modelling the regulation of CO and FT mRNA.Figure 3Schematic of the role of GI in destabilising CDF1 through an FKF1-dependent and FKF1-independent mechanisms, with resulting effects on CO and FT mRNA abundance. CDF1 protein data and simulations in a 35S:3HA-CDF1 line (CDF1ox; black lines, open squares) and 35S:HA-CDF1;fkf1 mutant (CDF1ox;fkf1; blue lines, open squares) in LDs. Data from Imaizumi et al (2005). CDF1 protein data and simulations in a 35S:HA-CDF1 line (CDF1ox; as in B) and 35S:HA-CDF1;gi-2 mutant (CDF1ox;gi; red lines, open squares) in LDs. Plants were grown for 10 days in 16L:8D cycles. CO data and simulations in 16L:8D in WT (data: black lines, filled squares; simulation: black lines), the gi-2 mutant (data: red lines, open circles; simulation: dashed red lines) and fkf1 mutants (data: blue lines, open circles; simulation: dashed blue lines). Data from Sawa et al (2007). CO and FT mRNA data and simulations in LDs in WT (as in D, E) and the prr9;7 mutant (data: blue-green lines, open circles; simulation: dashed blue-green lines). Data from Nakamichi et al (2007). CO and FT mRNA data and simulations in 10L:14D in WT (as in D) and the cca1;lhy mutant (data: green lines, open circles; simulation: dashed green lines). Data from Nakamichi et al (2007).
Figure 2(A) μCT analysis of the femurs of 10-week-old male mice upon exposure to low oxygen concentration (14% pO2, n = 5) or ambient air (n = 7), and of 10-week-old Hif1aRank-/-; Hif2aRank-/- male mice upon exposure to low oxygen concentration (14% pO2, n = 6) or ambient air (n = 5) for 10 days (axial view of the metaphyseal region). Scale, 0.5 mm. (B) Histological analysis of the proximal tibias of 10-week-old male mice upon exposure to low oxygen concentration (14% pO2) or ambient air for 10 days, and of 10-week-old Hif1aRank-/-; Hif2aRank-/- male mice upon exposure to low oxygen concentration (14% pO2 ) or ambient air (toluidine blue staining [arrow, osteoclasts]). Scale, 100 μm. (C) Parameters for osteoclasts and osteoblasts during bone morphometric analysis of 10-week-old male mice under low oxygen concentration (n = 3) or ambient air (n = 5), and 10-week-old Hif1aRank-/-; Hif2aRank-/- male under low oxygen concentration (n = 3) or ambient air (n = 5) for 10 days.
Figure 6Schematic showing the relationship between a signal (grey) and the corresponding output (red). A continuous signal pulse leads to maximal output (top left) whereas well-separated pulses independently increase output (top right). When pulses are separated by shorter periods when signals persist, the combined output is increased between these two bounds (bottom row). Single pulses of varying duration were applied to OptoCAR‑T cells and the geometric mean of GFP expression was measured for all pulse lengths 24 hours after initiation of signaling, with illumination started once the signal pulse had ended. All values have been corrected for background GFP fluorescence from non-activated OptoCAR‑T cells and shown relative to the output at 24 hours. A representative dataset is provided in Figure EV4F. Bars show mean ± SEM (n=3) Equivalent datasets as in (B) for the geometric mean of CD69 upregulation. A representative dataset is provided in Figure EV4F. Bars show mean ± SEM (n=3) Schematic illustrating the pulse-frequency modulation of signal pulses in OptoCAR‑T cells. For a given pulse duration, the length of the light interval between pulses was varied between duty cycles of 100% (continuous) and 25%. The output after 24 hours was measured for all samples. Series of experiments showing how NFAT-mediated GFP expression is modulated by pulsatile trains of signaling. A combined signaling period of 6 hours was broken into signal pulses ranging from 15 minutes to 2 hours and the light-induced 'off' interval between these pulses varied, denoted as x in the schematic above each plot. Geometric mean of GFP intensity is shown plotted as a function of light-induced 'off' interval, relative to a single, continuous 6-hour output. Bars show mean ± SEM (n=3) of biological replicates. Series of experiments showing how CD69 upregulation is modulated by pulsatile trains of signaling, from same dataset as in (E). Bars show mean ± SEM (n=3).