EMBO/sd-character-level-ner huggingface dataset:
Figure 4Representative confocal z-stack images of microglial lysosomes (CD68, green) in the IC (inferior colliculus) of control and Cdc50a cKO mice. Scale bar, 20 μm. Representative confocal z-stack and colocalization images of Gephyrin (green), IBA1 (blue), and CD68 (red) in the IC of control and Cdc50a cKO mice. Scale bar, 20 μm. Bar graphs showing the relative colocalization area of Gephyrin and CD68 in the IC of control and Cdc50a cKO mice (n = 3 for each group, **P = 0.0024). Schematic diagram of microglial ablation in control and Cdc50a cKO mice using DMSO- or PLX3397-containing chow. DMSO- or PLX33997-containing chow was administered for 2 weeks before oil or 4-OHT injection. Bar graphs showing the audiogenic seizure scores of Cdc50a cKO mice treated with DMSO or PLX3397 after a 20 kHz/90 dB auditory stimulus (n = 7 for Cdc50a cKO;DMSO and 3 for Cdc50a cKO;PLX3397). Representative confocal z-stack images of microglia (IBA1, green, upper) and inhibitory synapses (Gephyrin: green, VGAT: red, lower) in the IC of control;DMSO, control;PLX3397, Cdc50a cKO;DMSO and Cdc50a cKO;PLX3397 mice. The dashed line represents where the high magnified insert was taken from. Scale bar, 20 μm. Bar graphs showing the relative area of IBA1-positive cells in the IC of control;DMSO, control;PLX3397, Cdc50a cKO;DMSO and Cdc50a cKO;PLX3397 mice (n = 3 for each group, control;DMSO versus control;PLX3397, *P = 0.0428, control;DMSO versus Cdc50a cKO;DMSO, ***P = 0.0002, Cdc50a cKO;DMSO versus Cdc50a cKO;PLX3397, ***P = 0.0005). Bar graphs showing the number of inhibitory (Gephyrin- and VGAT-positive) synapses in the IC of control;DMSO, control;PLX3397, Cdc50a cKO;DMSO and Cdc50a cKO;PLX3397 mice (n = 3 for each group, P**** < 0.0001).
Figure 3A. Experimental design and examples of stretched DNA fibers with different patterns of DNA synthesis around the ICL lesion. Fibers were stained with anti-CldU (red), anti-IdU (green) and anti-Dig (magenta). Schematics over the fiber images highlight the position of the ICL (circle) in each replicative structure. Scale bar, 10 µm. B. PrimPol mutants used in the experiments. AxA is a PrimPol catalytic mutant. ∆Zn and CH are primase-null, polymerase-proficient PrimPol mutants. RBDm is a PrimPol mutant defective for RPA-binding. C. Histograms show the percentage of the different replication patterns observed in each experimental condition (average and SD of three assays). Circle dots in each column represent the values of individual replicates. Statistical analysis: two-way ANOVA followed by Bonferroni post-test comparing each condition to the WT. p-values of individual comparisons are indicated. Immunoblots showing the levels of V5-PrimPol proteins (WT and mutant derivatives). Ponceau-S staining is shown as loading control. D. Experimental design of ICL-localized DNA fibers followed by S1 nuclease incubation. Hypothetical fiber containing ssDNA downstream of the ICL (circle) turns into a DSB after S1 nuclease action. This would lead to an increase in apparent stalled forks or shortening of IdU length downstream of the lesion. E. Histograms show the percentage of the different replication patterns observed in each experimental condition (average and SD of two assays). Circle dots in each column represent the values of individual replicates. Statistical analysis: two-way ANOVA followed by Bonferroni post-test. p-values of individual comparisons are indicated. F. Dot plot shows the distribution and median (horizontal red line) of IdU track length after an event of ICL traverse in each condition. Data pooled from two replicates are represented (n ≥100 cells per condition). Statistical analysis was conducted with Kruskal-Wallis test and Dunns post-test. p-values of individual comparisons are indicated. G. Histograms show the percentage of the different replication patterns observed in each experimental condition (average and SD of two assays). Circle dots in each column represent the values of individual replicates. Statistical analysis: two-way ANOVA followed by Bonferroni post-test. p-values of individual comparisons are indicated. H. Immunoblots showing the levels of BLM, FANCD2 and PrimPol after BLM or FANCD2 downregulation in WT and PRIMPOL KO cells for experiments in (G). Ponceau-S is shown as loading control.
Figure 3. Transcriptional activity of subtelomeric p53-response elements. (A) Luciferase test constructs containing either empty test vector (top), p53-response elements from 18q subtelomere (middle) or MDM2 promoter (lower) were assayed in HCT p53-/- cells transfected with two independent preparations of empty vector or p53 WT expression vector. (B) RT-qPCR measure of p21 (top panel) or PARD6G (lower panel) in HCT116 p53-/- or p53+/+ cells after treatment with DMSO or 50 µM etoposide. (C) RT-qPCR analysis of TERRA transcripts from subtelomere 13q (upper) or 2q (lower). (D) RT-qPCR analysis of TERRA transcripts from 18q using two independent primer sets pr1 (top panel) or pr2 (lower panel) from HCT116 p53+/+ cells cultured with serum (grey bars) or in HBSS lacking serum (black bars). Error bars represent SD and * indicates p value <.05 using student t-test comparing treated and untreated samples.
Up-regulation of Wee1 kinase compensates constitutive Cdk1 and Cdc25C activation in G2/M phase after CLU silencing.Figure 7(A) Western blot for CLU, Cdk1 Y15, Cdk1 and Wee1 in PC-3 cells after siSCR or siCLU transfection followed by synchronization and nocodazole release at indicated time points. Vinculin was used as loading control (B) Wee1 expression level was evaluated in PC3 cells after CLU silencing by real-time PCR. Error bar represent mean ± SEM, n=3, *** p<0.001 (p=0.0001) by unpaired t-test followed by Welch's correction. (C) FACS analysis showing percentage of the PC3 cells positive for phosphohistone H3 after transfection with siSCR or siCLU in presence and absence of Wee1 inhibitor, MK-1775. Error bar represent mean ± SEM, n=3, ****p<0.0001 by Mann-Whitney (D) Western blot for CLU, Wee1, Cdk1 Y15, Cdk1 and Cyclin B1 in PC3 cells after transfection with siSCR or siCLU followed by synchronization and treatment with or without MK-1775, a specific Wee1 inhibitor, for 12 hours. Cells were harvested at different time points after nocodazole release. Vinculin was used as loading control.
Figure 7A. MCF10A cells expressing shRNA against AMOTL2 were complemented with shRNA-resistant Flag-AMOTL2 WT or the K347/408R mutant. The indicated cells were harvested and analyzed by Western blotting for the indicated proteins. B. mRNAs isolated from cells in (A) were analyzed by RT-qPCR against indicated EMT marker genes (n=4). Error bars indicate the S.E.M. (*p< 0.05, **p < 0.01, ***p < 0.001; paired Student's t-test). C. Transwell migration assay was performed using cells in (A) (n=8). We analyzed 5 random fields for each sample. Error bars indicate the S.E.M. (**p< 0.01, paired Student's t-test). D. Representative images of soft agar assay results using cells in (A). Scale bar, 1 mm. E. Soft agar assay was performed using cells in (A) (n=8). Error bars indicate the S.E.M. (**p < 0.01, ***p < 0.001, paired Student's t-test).
Figure 1 A MMS-induced checkpoint activation is not altered in irc5-Δ1 cells. The level of Rad53 phosphorylation was analyzed in wild type (W303-1a) and irc5-Δ1 (IL012) cells by Western blot with an anti-Rad53 antibody  B Prolonged checkpoint activation in irc5-Δ1 cells during recovery from MMS treatment. Rad53 phosphorylation status was analyzed in wild type (W303-1a) and irc5-Δ1 (IL012) cells by Western blot with an anti-Rad53 antibody; M, MMS  C Lack of Irc5 results in an elevated number of cells with Rad52-YFP foci. At indicated time points samples of wild type (W3749-14C) and irc5-Δ1 (TB039) cells were collected, washed and processed for microscopic analysis. Error bars represent mean value ± standard deviations of mean (n=5). Unpaired t-test was used to calculate the P-value. Representative microscopic photos of cells are shown. Scale bars: 5 μm  D Disruption of IRC5 leads to accumulation of ssDNA-containing lesions. ChEC analysis of wild type (BYR52MN) and irc5-Δ1 (TB040) cells cultured in the presence or absence of 0.03% MMS for 2 hours (n=3)  E Quantification of data presented in D (n=3) F Increased levels of bright Rfa1-YFP foci in cells lacking Irc5. At indicated time points culture samples of wild type (W3775-12C) and irc5-Δ1 (TB041) cells were collected, washed and processed for microscopic analysis. Error bars represent mean value ± standard deviations of mean (n=5). Unpaired t-test was used to calculate the P-value. Representative microscopic photos of cells are shown. Scale bars: 5 μm
Figure 7Upon binding of IL-17 to IL-17RA and IL-17RC, 6 molecules of ACT1 are recruited, creating docking site for trimeric TRAF6. TRAF6-created K63-polyubiquitin linkages promote activation of signaling pathways, but also trigger recruitment of TBK1 and IKKε kinases to IL-17RSC. Both kinases then phosphorylate ACT1 in disordered region of the protein. This leads to a spatial separation of TRAF6-binding sites present on ACT1, decreased avidity for TRAF6 and its release from IL-17RSC, ultimately leading to inhibition of signaling. In addition, TBK1/IKKε enable recruitment of TRAF2 in kinase-activity independent manner to promote stabilization of mRNA of target cytokines.
Characterization of Netrin-3 in SCLC.Figure 4A. Deep analysis of netrin-3 gene expression in lung cancer cell lines (n=76; RNA-Seq). B. Q-RT-PCR analysis of netrin-1 and -3 expression, in lung cancer cell Lines (Error bars indicate SD, n=3).  C. Representative netrin-3 and -1 mRNA detection, using RNAscope on SCLC paraffin embedded tumor sections. Negative control DAPB, positive control PPiB. Each brown dot is a unique molecule of mRNA of each targeted gene (n=10, scale bars 20µm). D. Quantification of netrin-3 and -1 expression in human SCLC paraffin embedded tumor sections (High≥ 50% marked cells; Low≤ 5%).
Figure 7A. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF or lncRNA-MIF-AS as indicated. Forty-eight hours after infection, growth curves were measured for the indicated periods of time. Data shown are mean ± SD (n = 3). B. HeLa cells were infected with lentiviruses expressing control shRNA, lncRNA-MIF shRNA-1, -2 or -3. Forty-eight hours after infection, growth curves were measured for the indicated periods of time. Data shown are mean ± SD (n = 3). C. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF or lncRNA-MIF-AS as indicated. Forty-eight hours after infection, cells were stained with PI and analyzed by flow cytometry to identify phases of cell cycle. D. HeLa cells were infected with lentiviruses expressing control shRNA, lncRNA-MIF shRNA-1, -2 or -3. Forty-eight hours after infection, cells were stained with PI and analyzed by flow cytometry to identify phases of cell cycle. E. The percentage numbers of cells (Fig 7C) in G1, S or G2/M phase were analyzed by FlowJo 7.6 software. Data shown are mean ± SD (n = 3). F. The percentage numbers of cells (Fig 7D) in G1, S or G2/M phase were analyzed by FlowJo 7.6 software. Data shown are mean ± SD (n = 3).
A heterozygous Y223C caPOT1 mutation does not induce a DNA damage response at telomeres, but elongates telomeres A) Schematic for the "scarless loopout" strategy used to generate POT1WT/Y223C in hESCs.Figure 3A) Schematic for the "scarless loopout" strategy used to generate POT1WT/Y223C in hESCs. B) TIF analysis of wild-type, POT1WT/Y223C hESCs, and the indicated controls using antibodies against TRF1 and γ-H2AX. Scale bars = 10µm. Lentiviral expression of TPP1∆PBD served as a positive control to demonstrate that cells have an intact telomere DNA damage response. TPP1∆PBD infected cells were analyzed 72h post infection and without selection. C) Box and whisker plot (showing min, max and average) quantifying the fraction of TIF-positive telomeres per cells as shown in (B) (two-tailed Mann-Whitney test, one biological replicate). D) STELA showing telomeres of POT1WT/Y223C, POT1WT/Puro, and an untargeted control hESC clone that was isolated and cultured in parallel to the edited clone. E) Quantification of two STELA experiments, including that shown in (D). Indicated in red is the mean telomere length and the error bars indicate the standard deviation.
4D7 has no effect on normal progenitor cells and shows activity against ruxolitinib-resistant cells without haematological toxicity.Figure 4A. Graph showing effect of ruxolitinib on megakaryocyte differentiation at 50 and 100 nM in healthy CD34+ cells (n= 4 different cords collected with 3 technical replicates). Data information: Error bars indicate standard error of mean. All P values are unpaired Student's unpaired t-test. B. Effect of 4D7 on TPO-dependent megakaryocyte differentiation of healthy CD34+ cells cultured with 20 µg/mL 4D7 or IgG control (n= 2 biological replicates, with 2 technical replicates). Data information: Error bars indicate standard error of mean. C. Total numbers of hematopoietic colonies plated in MethoCult from healthy cord blood after treatment with 4D7 or IgG. CFU-GM, colony forming unit- granulocyte macrophage; BFU-E, blast forming unit-erythroid; CFU-GEMM, colony forming unit granulocyte, erythroid, monocyte, megakaryocyte (n=3 biological replicates). Data information: Error bars indicate standard error of mean. D. CD41+ megakaryocyte colonies from healthy CD34+ cells observed on collagen matrix after 4D7 treatment. Colonies were cultured in TPO, SCF, IL-9 and IL-3. Scale bar indicates 100µm. E. Number of megakaryocyte colony forming units observed in MegaCult assay after treatment of healthy cord blood CD34+ cells with either IgG or 4D7 (n=3 biological replicates). Data information: Error bars indicate standard error of mean. F. Western blot showing signalling in ruxolitinib-resistant TF-1 TpoR CALRdel61 compared to ruxolitinib sensitive TF-1 TpoR CALRdel61 cells after treatment with 100nM ruxolitinib for 16 hours or 30 minutes and blotted for phospho-STAT5, total STAT5, phospho-ERK, total ERK and actin as indicated. Ruxolitinib-sensitive cells were non-viable after 16 hours of treatment. G. Comparison of cell growth after DMSO, 20 µg/mL 4D7, IgG or 100 nM ruxolitinib treatment over 4 days of ruxolitinib-resistant TF-1 TpoR CALRdel61. Cells counted using Trypan Blue exclusion (n=3 biological replicates with 3 technical replicates). H. Number of colonies observed from cells plated in MethoCult following 72 hours of treatment with DMSO, 20 µg/mL 4D7, IgG or 100nM ruxolitinib performed in ruxolitinib-resistant TF-1 TpoR CALRdel61 cells (n=3 biological replicates with 3 technical replicates). Data information: Error bars on represent standard deviation. All P values are unpaired Student's unpaired t-test.
Figure 2A) Representative images of mt-Keima expressing spinal cord sections of SOD1-G93A and Non Tg mice at 90 days. Mt-Keima fluorescence with excitation at 458nm is pseudocolored in green and at 543nm is pseudocolored in red. Emissions were recorded sequentially at 600-650nm. Scale bar, 150 µm. B) Representative images of mt-Keima expressing motor neurons in the ventral horn of SOD1-G93A and Non Tg mice at 90 days, imaged and pseudocolored Scale bar, 20 µm. C) Quantification of the rate of mitophagy in motor neurons expressed as the percentage of area with high ratio (543/458nm) signal normalized by the total mitochondria area. Data were collected from mice at 60 days (n= 3, 1 male and 2 females), 90 days (n= 4, 2 males and 2 females), and 120 days (n= 4, 2 males and 2 females). Number of neurons imaged: 60 days, n= 77 Non Tg and 87 SOD1-G93A; 90 days, n= 118 Non Tg and 173 SOD1-G93A; 120 days, n= 110 Non Tg and 130 SOD1-G93A. Comparisons within pairs showed that mitophagy is increased in SOD1-G93A spinal cords at 60 and 90 days compared to Non Tg. Results are expressed as mean ± SEM. For 60 days, **p= 0.0022; for 90 days, ***p= 0.0001; for 120 days, p= 0.094, all by unpaired Mann-Whitney test. Comparisons along time showed that mitophagy increases over time in both genotypes. For Non Tg, by unpaired Kruskal-Wallis's test with Dunn's correction, **p= 0.0046 and ***p= 0.0001. For SOD1-G93A, by unpaired Kruskal-Wallis's test with Dunn's correction, **p=0.0077 and *** p= 0.0001.
Association of autism severity readouts in the discovery and 3 independent replication samples with the number of proautistic genotypes in the 8SNP risk model derived from the broader fragile X gene family.Figure 4(A) PAUSS (PANSS autism severity score) composition and item intercorrelation pattern in the GRAS sample of male schizophrenic individuals (discovery sample). Cronbach's alpha is presented as measure of internal consistency and also provided for the male replication samples I and II. (B) Association of PAUSS with the number of proautistic genotypes of the 8SNP risk model in the discovery sample. (C) PAUSS comparison of extreme groups with high and low numbers of accumulated proautistic genotypes in the discovery sample; binary logistic regression analysis with non-z-standardized PAUSS as dependent variable; mean±SEM. (D) PAUSS comparison of extreme groups with high and low numbers of accumulated proautistic genotypes in replication sample I of male schizophrenia patients; binary logistic regression analysis; mean±SEM. (E) PAUSS comparison of extreme groups with high and low numbers of accumulated proautistic genotypes in replication sample II of male disease control patients; Mann-Whitney U test; mean±SEM. F) The highly significant correlation of PAUSS and social support underlines the validity of social support as an autism proxy phenotype; mean±SEM. (G) Comparison of the extreme groups with high and low numbers of accumulated proautistic genotypes for the autism proxy phenotype social support in the discovery sample; binary logistic regression analysis; mean±SEM. (H) Comparison of the extreme groups with high and low numbers of accumulated proautistic genotypes for the autism proxy phenotype social support in the male replication sample III from general population; binary logistic regression analysis; mean±SEM. For all replications p-values for one-sided tests are shown.
UBE2QL1 translocates in the vicinity of damage sensors, ubiquitination targets and ubiquitin effectors at permeabilized lysosomes.Figure 4Immuno-electron microscopy of LLOMe-treated HeLa cells. Cells in (A) overexpress mCherry-Gal3 and a dominant-negative C160S mutant of GFP-YOD1, and were immunostained for p62 (A) p62 (10 nm gold particles) localizes as a cap (arrowheads) to the outer limiting membrane of lysosomes (L). Scale bars: 200 nm. Immuno-electron microscopy of LLOMe-treated HeLa cells. Cells in (B) overexpress mCherry-Gal3 and a dominant-negative C160S mutant of GFP-YOD1, and were immunostained for p62 and K48. (B) K48 ubiquitin chains (15 nm gold particles, arrows) are found in the lumen of lysosomes, that also contain p62 (10 nm gold particles, arrowheads) at their outer membrane. Scale bars: 200 nm. Immuno-electron microscopy of LLOMe-treated HeLa cells. (C) Overexpressed UBE2QL1-GFP (10 nm gold particles) is found in late endosomes (LE, Inset) and lysosomes. M = mitochondrion. Scale bars: 200 nm. D, E Comparative UBE2QL1 proximity mapping before and after lysosome damage using SILAC mass spectrometry. HeLa cells expressing a UBE2QL1-APEX2 fusion were EtOH (light-labelled, L)- or LLOMe (heavy-labelled, H)-treated and pulsed with biotin phenol (30 min) and H2O2 (1 min). Proteins detected in replicative streptavidin purifications are depicted in a Venn diagram (D) and displayed high correlation coefficients (E). , F Comparative UBE2QL1 proximity mapping before and after lysosome damage using SILAC mass spectrometry. HeLa cells expressing a UBE2QL1-APEX2 fusion were EtOH (light-labelled, L)- or LLOMe (heavy-labelled, H)-treated and pulsed with biotin phenol (30 min) and H2O2 (1 min). (F) Results from the four experiments are summarized in a volcano plot. Horizontal dotted line represents the significance threshold (p > 0.05). The vertical lines indicate the fold change cutoff (log2 (H/L) ≥ 1.5). Red dots indicate proteins above the significance and fold-change thresholds. Protein names are color-coded in blue (endosome/lysosome-associated), green (endolysosomal damage/autophagy-associated) and orange (VCP/p97 and its cofactors).
a) Quantification of Atg8a proteins levels from head extracts normalized to α-tubulin (n = 17 or 8–9 technical replicates from two independent biological aging replicates for all data, F = 7.figf4(a) Quantification of Atg8a proteins levels from head extracts normalized to α-tubulin (n = 17 or 8-9 technical replicates from two independent biological aging replicates for all data, F = 7.52, one-way-ANOVA with Tukey post-test). The full-length blot is shown in Supplementary Figure 9a. (b) Western blot analysis of poly-ubiquitinated proteins (SDS-soluble protein fraction) from head extracts from Spd− (3 and 30 d old) flies compared with Spd5mM+ (3 and 30 d old) flies. Right, quantification of poly-ubiquitinated proteins levels normalized to α-tubulin (n = 10 or 5 technical replicates from two independent biological aging replicates for all data, F = 6.32, one-way ANOVA with Tukey post-test). The full-length blot is shown in Supplementary Figure 9b. (c-f) Adult brains of 3-d-old and 30-d-oldSpd− flies, as well as 3-d-old and 30-d-oldSpd5mM+ fliesimmunostained for ref(2) P. Scale bar represents 25 μm. (g) Quantification of ref(2) P intensity in the central brain region normalized to 3-d-oldSpd− (n = 12-13 independent brains for all data, F = 26.58, one-way ANOVA with Bonferroni correction). *P 0.05, **P 0.01, ***P 0.001; P > 0.05. Data are presented as mean ± s.e.m.
A-type minor introns are spliced less efficiently.Figure 6Density plots showing intron retention (Ψ) values in the HeLa unedited parental control cell line (depleted of GAPDH) of A- and G-type minor introns that were either unchanged or retained after CENATAC depletion (top) or ZRSR2 mutation (bottom). The median psi-values of the retained G- and A-type introns are significantly higher (Psi=0.130 and Psi=0.115, respectively; P<0.01, Mann-Whitney Rank Sum Test) compared to the unchanged introns (Psi=0.081) in the CENATAC depletion dataset. RT-PCR P120 reporter assay(Hall & Padgett, 1996) to measure the relative usage of A-type (AT-AC) and G-type (GT-AG) 5' splice sites in direct competition. Upper: schematic diagram showing the overall architecture of the reporter construct with its down- and upstream splice site (thick red and blue bars, respectively) and the products created by splicing (Dn and Up, respectively). Lower: RT-PCRs of the reporter with A- or G-type splice sites in the down- or upstream positions as indicated below the gel. SS, splice site. *, PCR product after use of a cryptic major splice site (not shown in the schematic). Quantification of relative splice site usage of A- and G-type splice sites in (B) (three biological replicates).
Figure 7(A B) Neutrophil recovery and morphologic differentiation were assessed in BM (panel A) and PB (panel B) of CCIN mice surviving on day 9. White arrows indicate neutrophil nuclear segmentation. (C) PBneutrophil production and nuclear segmentation were assessed in control and Am80-GCSF mice surviving on day 13 (section i). (C) PBneutrophil production and nuclear segmentation were assessed in control and Am80-GCSF mice surviving on day 13 (section ii). (C) The increased spleen size on day 9 in Am80-GCSF mice dropped to the levels similar to those in control mice on day 13 (section iii). (C) The increased spleen weight on day 9 in Am80-GCSF mice dropped to the levels similar to those in control mice on day 13 (sections iii, iv). (D) Significant loss of body weight occurred in GCSFmice on day 9. (E) Ultrastructure analysis of neutrophils collected from mice surviving on day 11. Neutrophil from GCSFmice showed less granules (section i, images 1 vs. 2 or 3), more intracytoplasmic vacuoles (sections i, ii), and inner nuclear membrane dilation (sections i, iii). Pg: primary granules; Sg: secondary granules.