EMBO/sd-character-level-ner huggingface dataset:
Figure 2(A-C) Rescue of repression of poly-GA and repeat RNA by wild type (wt) hnRNPA3 and hnRNPA2 but not by the RNA binding mutant hnRNPA3DxD. (B) n=3 experiments performed in duplicates; (C) n= 2 experiments performed in duplicates. ANOVA, Tukey. (D-F) Rescue of repression of poly-GA and repeat RNA by hnRNPA3WT but not the M9-NLS deletion mutant. (E) n=3 experiments performed in duplicates; (F) n=4 experiments performed in duplicates. ANOVA, Tukey. All Graphs are shown as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001 (See also Figure EV1).
Figure 1Figure 1. Identification of non-canonical p53 binding sites in human subtelomeres. (A and B) ChIP-seq tracks for p53 in camptothecin (CPT) treated or untreated HCT116 cells were aligned with RNAPII, CTCF, RAD21, and SMC1 for human chromosome18q (A) or 13q (B) subtelomeres. Position relative to the centromeric end of the terminal TTAGGG repeat tract are indicated in bp at the top. Subtelomeric DNA paralogy segment tracks, internal telomere-like sequence tracks (red CCCTAA), and annotated RefSeq gene tracks are indicated above the ChIP-seq tracks. (C) Alignment of human subtelomeric elements containing non-canonical p53 recognition sequences. The position of the RepeatMasker-defined LTR10c element is shown; also note the (TG) n microsatellite repeat adjacent to the p53 binding site. (D) Schematic organization of non-canonical p53 binding sites found in human subtelomeres.
Figure 5A. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF or lncRNA-MIF-AS. By Forty-eight hours infection, cells were treated with MG132 or DMSO (vehicle) for 6 h. Cell lysates were then analyzed by Western blotting with the indicated antibodies. B. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF or lncRNA-MIF-AS. Forty-eight hours after infection, total RNA was subjected to analyzing the levels of lncRNA-MIF and lncRNA-MIF-AS by real-time RT-PCR. Data shown are mean ± SD (n = 3; **P < 0.01, two-tailed t-test). C. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF or lncRNA-MIF-AS. By 48 h infection, total RNA was subjected to real-time RT-PCR analysis. Data shown are mean ± SD (n = 3; **P < 0.01, two-tailed t-test). D. HeLa cells were infected with lentiviruses expressing control shRNA, lncRNA-MIF shRNA-1, -2 or -3. By 48 h infection, cells were treated with MG132 or DMSO for 6 h. Cell lysates were then analyzed by Western blot analysis with the indicated antibodies. E. HeLa cells were infected with lentiviruses expressing control shRNA, lncRNA-MIF shRNA-1, -2 or -3. Forty-eight hours after infection, total RNA was subjected to real-time RT-PCR analysis. Data shown are mean ± SD (n = 3; **P < 0.01, two-tailed t-test).
TRaMs result in elevated enzyme expression following xenotransplantation.Figure 2A. Schematic of the transplant protocol optimized for T-Rapa cells. NOD/SCID/Fabry (NSF) mice were conditioned and TRaMs were administered intravenously - details are indicated in Materials and Methods. Mice were euthanized 4 weeks post-infusion. B. Engraftment was evaluated by flow cytometry for human CD3 expression. C, D. α-Gal A activity in plasma, and specific activities in liver, spleen, heart and kidneys were measured after transplant of transduced healthy donor (HDo)-derived (C) or Fabry donor (FDo)-derived (D) TRaMs, non-transduced (NT) cells, or in sham-treated NOD/SCID (NS) and NSF mice.
Figure 5Immunoblotting analysis of signalosome components by immunoblotting in BCL9, CLTC or in both BCL9 and CLTC knockdown mitotic cells with CHX. Quantification of the stability of several LRP6 signalosome components by immunoblotting of mitotic cells with CHX. Immunofluorescence confocal analysis of Ser1490-phosphorylated LRP6 and CLTC co-localization in mitotic HeLa cells; white arrows indicate the colocalization. Scale bars represent 5 μm. Immunofluorescence analysis of α-tubulin, Axin1 and CLTC triple-staining in mitotic cells. The white arrows indicate the colocalization. Scale bars represent 5 μm. Immunofluorescence analysis of colocalization of LRP6CAGFP and CLTC or CD63 in interphase cells. The white arrows indicate the colocalization. Scale bars represent 5 μm. Quantification of signalosome components and mitotic Wnt target expression level by immunoblotting in natural mitotic cells treated for 1 hour with DMSO or MDC, an inhibitor of clathrin-mediated-endocytosis. A representative image of soft agar oncogenic colony formation assays for stable knockdown cells. A representative image of the mouse xenograft tumours for stable knockdown cells at the end point of the experiment.
Effects of MitoPQ treatment in the absence or presence of APOPT1 A Western blot analysis of SDS-PAGE of different mitochondrial proteins in total lysates from the indicated cell lines treated with 5 µM MitoPQ at the indicated times.Figure 7 A Western blot analysis of SDS-PAGE of different mitochondrial proteins in total lysates from the indicated cell lines treated with 5 µM MitoPQ at the indicated times. UT: untreated cells. B Densitometric quantification of APOPT1GFP signal during the treatment in two biological replicas. C Densitometric quantification of MT-CO1 signal in the non-complemented APOPT1-less cells (S6 GFP) vs. the complemented cells (S6 APOPT1GFP). Three biological replicas (n=3) were carried out for each cell line. The signals in UT S6 APOPT1 were considered 100%. The levels of MT-CO1 were significantly lower in the S6 GFP patient cells after 20 hours of MitoPQ treatment compared to the untreated cells (*P = 0.0202, two-tailed unpaired Student's t-test). D Densitometric quantification of MT-CO2 signal in the non-complemented APOPT1-less cells (S6 GFP) vs. the complemented cells (S6 APOPT1GFP). Three biological replicas were carried out for each cell line. The signals in UT S6 APOPT1 were considered 100%
Figure 4(A) KPC tumor growth delay after treatment with either 12 Gy (day 0), anti-PD-L1 (days 0, 3, 6 and 9; black arrows), anti-CD8 alone (days 0 3, 6 and 9) or their combinations, as indicated. (B) KPC tumor growth delay after treatment with either 5x3 Gy (day 0-4), anti-PD-L1 alone (days 0, 3, 6 and 9; black arrows), anti-CD8 alone (days 0, 3, 6 and 9) or their combinations, as indicated. (C) KPC tumor growth delay after either simultaneous (day 0, 3, 6 and 9; black arrows-upper row) or sequential (day 6, 9, 13 and 16; black arrows-lower row) addition of anti-PD-L1 to RT (day 0). The average time (days) for tumors to reach a volume of 400 mm3 from day 0 for Figure 4A-C is also shown (means ± SD, n=1, One-way ANOVA, Bonferroni test). No weight loss was observed in the in vivo experiments.
Figure 4A Diagram of the mating type loci in S. pombe. The position of the silencing reporter gene (kint2::ura4+) is shown. The target regions in the ChIP analysis are indicated by black bars above (kint2::ura4+) and below (cenH) in the diagram. B Heterochromatic silencing assays of wild-type and mutant Hht1-FH strains. Silencing at the mating-type Kint2::ura4+ was evaluated. Ten-fold serial dilutions of the indicated strains were spotted onto non-selective medium (N/S), medium lacking uracil (-Ura), and medium containing 5-FOA (5-FOA). C ChIP analysis of H3K9me2 levels associated with the mating-type loci, relative to the control act1+ locus. Error bars indicate standard errors from three technical replicates. D ChIP analysis of Rik1-myc levels associated with the mating-type cenH and centromeric dg loci, relative to the control act1+ locus. Error bars indicate standard errors from three technical replicates. E Heterochromatic silencing assays comparing the effects of H3 lysine substitution mutants. Silencing at the mating-type kint2::ura4+ was evaluated
Figure 1(a) Workflow schematic of BOEC generation from PBMC samples isolated from NAFLD patients and healthy donors. (b) Upper panel: Representative images of BOEC colonies from PBMC cultivation 10 days post-seeding. Dotted line in each image outlines a BOEC colony. Lower panel: Stabilized BOEC cultures after passaging of colonies (scale bar, 250 µm). (c) Proliferation doubling time for NAFLD and healthy BOECs based on passages 2 and 3. Sample sizes are n = 9 healthy, n = 7 NAFLD; with 3 biological replicates per donor. Box-whisker plots indicate median (middle line), 25th, 75th percentile (box) and the lowest/ highest data points (whiskers). ns, not significant (t-test). (d) Left panel: Flow cytometry characterization of BOECs for endothelial, leukocyte and progenitor cell markers (grey - isotype control; red/blue/green/purple - cell lineage marker staining). HUVEC and monocytic THP-1 cells were used as positive controls for endothelial and leukocytic markers respectively. Right panel: Percentages of positively expressing cells. Sample sizes are n = 4-6 healthy, n = 2-7 NAFLD. (e) Representative images of immunostaining for endothelial markers, PECAM1 and CDH5 (scale bar, 100 µm). (f) Left panel: Longitudinal monitoring of BOEC angiogenesis in fibrin gel bead-based sprouting assays over 24h (representative images; scale bar, 100 µm). Right panel: Box-whisker plots of quantifications of sprout lengths and numbers of sprout per bead at 24h. n = 3 healthy, n = 3 NAFLD, with 4-5 beads analyzed per donor and 5-11 sprouts per bead.
Figure 6A Biochemical fractionation into RIPA-soluble (S) and RIPA-insoluble (I) fractions to analyze solubility of the different myc-TDP-43 variants (Wt, 12D and 12A) expressed in HeLa cells for 48 h. TDP-43 was detected by TDP-43 Western blot (upper blot, rabbit anti-TDP-43 C-term, Proteintech) and Myc Western blot (lower blot, mouse anti-Myc 9E10). B Quantification of myc-TDP-43 variants (Wt, 12D and 12A) in (S) versus (I) fractions extracted from TDP-43 Western blots of 4 independent replicates ± SD, plotted as S/(S+I). *p < 0.0332 by one way ANOVA with Dunnett´s multiple comparison test to Wt. C RIPA-insoluble material of the indicated EGFP-tagged TDP-43 variants (± NLS mutation) expressed in primary cortical neurons analyzed by filter-trap assay. D Primary hippocampal neurons expressing EGFP-TDP-43 Wt, 12D or 12A with additional NLS mutation. Bar, 80 µm. Right: Zoomed images of white squares (TDP-43 signal). Bar, 10 µm. E SG recruitment of EGFP-TDP-43 NLS mutant variants (Wt, 12D, 12A) in primary hippocampal neurons. SG formation was induced by 1h heat shock at 42°C. SGs and TDP-43 were monitored by G3BP1 antibody staining and EGFP fluorescence, respectively. For clarity, signals were converted to grey values in the individual channels (first two columns). In the merge (third column), EGFP-TDP-43 shown in green, G3BP1 in red and nuclei (DAPI staining) in turquoise. Bar, 20 µm.
The GABARAP/GATE‐16 protein subfamily is essential for autophagy.Figure 2(A) HeLa cells stably expressing GFP‐GATE‐16 (left panel) or GFP‐LC3B (right panel) were transfected with either non‐targeting siRNA (control siRNA) or a pool of GABARAP siRNAs (GABARAP, GABARAPL1, and GATE‐16) using DharmaFect reagent. After 72 h interval, the cells were incubated for 2 h in EBSS medium in the absence or presence of 0.1 μM Baf A and subjected to western blot analysis after lysis with RIPA extraction buffer. (B) Quantification of relative p62 and GFP‐LC3B level (upper panel) and degradation (lower panel) were performed as described in 'Materials and methods'. Mean±s.d. of three independent experiments is presented. (C) HeLa cells stably expressing GFP‐LC3B were transfected with non‐targeting siRNA or a pool of GABARAP siRNAs and treated as in (A). The cells were subjected to immunostaining with anti‐p62 after fixation, and analysed by confocal microscopy. The GFP intensity of three independent experiments was measured as described in 'Materials and methods' (right panel). Scale bar: 20 μm.
In vitro characterization of later passage ICL MEFs  (A) Growth curve of the later passage ICL lines Ch10 and Ch14 under adherent in vitro culture conditions (n=3 for each data set and error bars denote SD, p<0.Figure 6(A) Growth curve of the later passage ICL lines Ch10 and Ch14 under adherent in vitro culture conditions (n=3 for each data set and error bars denote SD, p<0.005 for Ch10 and p<0.0001 for Ch14). (See also Appendix Figure S3A) (B) Colony formation in ICL and control MEFs. 5000 cells were seeded in 10 cm plates and stained with Methylene blue after three days. Representative colony formation images in Ch10 and Ch14 are shown (left) and quantification of colonies formed (right) in the 4 ICL lines (n=3 for each data set and error bars denote SD, p<0.005 for Ch10, Ch12 and Ch14 and ns for Ch9). (C) Quantification of growth in ultra-low adherent, sphere forming conditions for the 4 ICL lines (n=3 for Ch10 and Ch14, n=6 for Ch9 and Ch12 and error bars denote SD, p<0.0001 for Ch9, p<0.001 for Ch10 and p<0.05 for Ch12 and Ch14). (D) Analysis of metabolites (glucose consumed, lactate produced and glutamine consumed) for the ICL lines. Spent media was analyzed after each ICL & control line was grown in culture for three days. Media in identical culture conditions, but with no plated cells, was used as a baseline for all samples.
a) ELISA of cytokines in supernatants of BMDMs incubated for 4 h after delivery of 3 μg DNase I, lactate dehydrogenase LDH (Control) or heat-inactivated DNase I (HI DNase I) and then stimulated with LPS, followed by stimulation for 1 h with ATP.figf5(a) ELISA of cytokines in supernatants of BMDMs incubated for 4 h after delivery of 3 μg DNase I, lactate dehydrogenase LDH (Control) or heat-inactivated DNase I (HI DNase I) and then stimulated with LPS, followed by stimulation for 1 h with ATP. (b) Quantitative PCR analysis of cytosolic mtDNA in BMDMs transfected as in a and then stimulated with LPS, followed by stimulation for 15 min with ATP. (c) ELISA of IL-1β secretion by Aim2+/+ and Aim2−/− BMDMs primed for 4 h with LPS (200 ng/ml), then transfected for 6 h with 1 μg mtDNA (through the use of liposomes as the vehicle), followed by stimulation for 1 h with ATP. *P 0.05, versus Aim2−/− cells treated with LPS and ATP (Student's t-test). (d) ELISA of cytokine secretion by LPS-primed Aim2−/− BMDMs transfected for 6 h with mtDNA, poly(dA:dT) or mtDNA predigested by DNase I (+ DNase; far left), followed by stimulation for 1 h with ATP. *P 0.05 (Student's t-test). Data are representative of three experiments (mean and s.d.).
Figure 5Conversion of the mmi-1 Model reaction network into influence diagrams based on two assumptions of kinetics. Left: symmetrical inhibition governed by balanced degradation rate constants in the complex. Right: asymmetrical and unidirectional inhibition governed by unbalanced degradation rate constants between mRNA and miRNA in the complex. Red: mRNA; Green: miRNA. Weights of the orange arrows describe the magnitudes of the degradation rate constants. Stability properties of 10000 randomly generated mmi-2 Model systems based on the procedure described in the legend to Figure 4C. Monostable systems (gray dots) and bistable systems (purple-green dots) are shown in the dimensions for a1/b1 and a2/b2. The color gradient denotes the range of bistability in terms of the control parameter. Orange dot denotes the condition under which degradation of mRNA and miRNA is balanced in both complexes. Red line is the threshold for bistability predicted by analytical methods. Cyan and magenta arrows serve as visual guides for panels C and D. Schematic depicting that when both complexes favor the same RNA species (mRNA or miRNA) with unbalanced degradation rate constants, the influences of mRNA and miRNA on each other are asymmetrical and unidirectional. Right diagram shows cancellation of two underlying feedback loops generated by following the inhibition arrows clockwise and counterclockwise. Schematic depicting a spectrum of influences from sponge-like inhibition to feedback-like mutual inhibition. When the influences of mRNA and miRNA on each other become cyclically directional, a feedback loop emerges. Four examples of the kinetic relationships (degradation rate constants) are shown in the box at right.
Figure 2A The model is represented as a process diagram and reactions are modulated by enzyme catalysis (circle-headed lines) or inhibition (bar-headed lines). Prefix 'p' represents phosphorylated species. Bold framed species were experimentally measured. White capsules represent inhibitors. Binding of the ligand Epo to its cognate receptor results in the phosphorylation of EpoR. Receptor and associated complexes are depicted in yellow, the AKT pathway is red, the ERK pathway is blue, and cell cycle genes and the S6 network is shown in green. B-G Model calibration with time-resolved quantitative immunoblot data of mCFU-E cells in blue and BaF3-EpoR cells in black. Growth-factor deprived mCFU-E cells (5x106 cells per condition) and BaF3-EpoR cells (1x107 cells per condition) were stimulated with different Epo doses and absolute concentrations were determined for pEpoR (B), pAKT (C), ppERK (D). The scale for pS6 (E) was estimated in arbitrary units. GTP-Ras (F) and ppERK were determined upon stimulation with indicated, color-coded Epo doses. pEpoR was analyzed by immunoprecipitation followed by immunoblotting, GTP-Ras was analyzed after pulldown using a fusion protein harboring GST fused to the Ras binding domain of Raf1 followed by detection by quantitative immunoblotting. For pAkt and ppERK, cellular lysates were subjected to quantitative immunoblotting. Calibrator proteins were used for EpoR, AKT, GTP-Ras and ERK to facilitate the conversion to nM concentrations. Experimental data represented by filled circles. Error bars represent standard deviation estimated by an error model. Solid lines represent model trajectories.
WASH and retromer complexes are required for SLC3A2 and Basigin sorting, and for full RAB21 activation and endosomal localization.Figure 7(A) Validation of WASH and retromer knockouts and SLC3A2 protein levels. Immunoblots of parental or FAM21, VARP and VPS29 knockout cell populations. (B) Antibody-uptake assays in parental or knockout FAM21, VARP and VPS29 cells. Cells were stained with an Alexa488 conjugated anti-mouse antibody. Arrowheads point to endosomal tubules. Data information: (B) scale bars represent 20µm (C) Percentage of cells with tubules in parental or in various knockout cells; SEM, n=3 independent experiments. Statistical tests used: (C) - Unpaired t-tests All error bars are SEM. Number of cells presented on each graph represents the total number of cells analyzed for all repeats. (D) Ratio of SLC3A2 integrated densities to GAPDH; SEM, n=3 independent experiments. Statistical tests used: (D) One sample T-tests All error bars are SEM. Number of cells presented on each graph represents the total number of cells analyzed for all repeats. Retromer is required for endosomal localization of RAB21. Immunofluorescence of transiently expressed GFP:RAB21 with endogenous (E) EEA1 in parental or VARP- and VPS29-deleted cells. Boxed region is magnified underneath. Data information scale bars represent 10µm or 2.5µm in enlarged views. Retromer is required for endosomal localization of RAB21. Immunofluorescence of transiently expressed GFP:RAB21 with endogenous (F) VPS26 in parental or VARP- and VPS29-deleted cells. Boxed region is magnified underneath. Data information: scale bars represent 10µm or 2.5µm in enlarged views. (G) Per cell Pearson Correlation between GFP:RAB21 and EEA1 or VPS26; SEM, n=3 independent experiments. Statistical tests used: (G) - Mann-Whitney tests. All error bars are SEM. Number of cells presented on each graph represents the total number of cells analyzed for all repeats.
Figure 7BTV-NS3 protein contains two conserved late domains PPRY and PSAP, which bind to ubiquitin E3 ligases and ubiquitin E2 respectively. BTV-NS3 is likely to interact directly with STAT2 and act as a scaffolding/adaptor protein that facilitates the transport of STAT2 for degradation. NS3 contains two lysine residues at position 13 and 15, which are K63 polyubiquitinated that target the NS3/STAT2 complex to the endosomes. STAT2 is targeted to autophagosome vesicles whose content is degraded after fusion with lysosomes. NS3 is probably deubiquitinated and allowed to recycle to the plasma membrane (as NS3 degradation was never observed).
CY harbors HDAC inhibitory activity that enhances nitrogen mustard's anticancer efficacy.Figure 2Upper panel, in vitro HDAC1 inhibition assay using bendamustine and CY. Lower panel, summary of CY IC50 against other HDACs. Upper panel, in vitro HDAC1 inhibition assay using bendamustine and CY. Lower panel, summary of CY IC50 against other HDACs. CY and CpdA, but not bendamustine or CpdB, inhibited HDAC activities in cancer cells. H1650 cells were treated with 20 μM CY, 20 μM CpdA, 200 μM CpdB, 200 μM Bend, or 10 μM SAHA for 12 h, and cell lysates were blotted for histone acetylation markers. Actin was used as a loading control. Molecular docking of CY with the structure of HDAC1/2 enzyme pocket. Alteration of CY side chain (Cpd B) caused loss of HDAC inhibitory activity and resulted in reduced ability to induce DNA damage. Cells were treated for 12 h. Cells treated with 200 μM Cpd B and 20 μM CY exhibited similar levels of γ-H2AX. Actin was used as a loading control. Summary of lethal dose 50 (LD50) of bendamustine, CY, Cpd A (HDAC inhibition only), and Cpd B (nitrogen mustard only) against 11 human cancer cell lines. Cpd A enhances the anticancer activity of bendamustine. H1650 cells were treated with various doses of bendamustine with or without 40 μM Cpd A (nontoxic dose, see Fig1C and Supplementary Fig S2) for 48 h and subjected to MTT viability assays.
The Atg12—Atg5–Atg16L1 complex regulates the stability of the ULK1–Atg13–FIP200 complex.Figure 2(A) Immunoblot analysis of Atg proteins in the indicated MEFs. (B) m5‐7 cells were cultured in regular medium containing 10 ng/ml Dox for 8 days, and then cultured in the absence of Dox for a further 2 days. (C, D) m5‐7 cells were cultured in regular medium with or without 10 ng/ml Dox for 4 days, and then cultured in the presence of 50 μg/ml CHX for the indicated time periods (C). mRNA levels of the indicated genes were measured by quantitative PCR following the 4‐day Dox treatment. Data represent mean±s.e. (*P0.05) (D). CHX, cycloheximide; Dox, doxycycline; MEFs, mouse embryonic fibroblasts; mRNA, messenger RNA; WT, wild‐type.
Figure 1(A) Transcription at GAL3 is visualized by addition of PP7 loops at the 5' of GAL3 in budding yeast. Example trace of the quantified fluorescence intensity of the transcription site. Traces are binarized to determine on (active) and off (inactive) times. (B) Histogram of GAL3 on time (burst duration) for cells with wt and mutated UAS, respectively, reveals shorter on times for mutated UAS. Errors indicate SE of 3 experiments, n = 324 cells UASwt, n = 250 cells UASmut. (C) Histogram of GAL3 off times. Errors indicate SE of 3 experiments. (D) cells were the UAS was mutated to a lower affinity UAS. (E). Average on time for UASwt and UASmut from exponential fit. Errors indicate SD of fit. (F) Average off time for UASwt and UASmut from exponential fit. Errors indicate SD of fit. (G) Example traces of quantified fluorescence intensity at the transcription site for 5'PP7 and 3'MS2 at GAL3. (H) Cross correlation of the PP7 and MS2 signals shows a peak at 37.9s±1.8s delay, n = 137 cells. Shaded area and errors indicate SEM. (I) Distribution of number of nascent RNAs at the transcription site determined by smFISH. The distribution of UASwt does not fit a Poisson distribution (grey line), supporting that GAL3 is not constitutively transcribed, but is transcribed in bursts. Example image in shown, yellow arrows indicate TSs. Scale bars: 5 μm. n = 11,839 cells. (J) for cells with UASmut. The distribution of UASmut fits a Poisson distribution, indicating that GAL3-UASmut is transcribed with random initiation of individual polymerases, similar to constitutive genes. n = 10,616 cells.