EMBO/sd-character-level-ner huggingface dataset:
Viral clusters contain specific nuclear body factors (A-C) Images of infected (right) or uninfected (left) ThP1 cells showing the vDNA (EdU) in red, the nucleus (DAPI) in blue, and selected nuclear body factors in green.Figure 3Images of infected (right) or uninfected (left) ThP1 cells showing the vDNA (EdU) in red, the nucleus (DAPI) in blue, and selected nuclear body factors in green. (A) Green image shows immunolabeling of CPSF6. (B) Green image shows RNA-FISH against NEAT1. (C) Green image shows immunolabeling of SC35. See also Appendix Figs. S9-14. (D) Boxplots show Pearson correlations r between vDNA (EdU) and CPSF6, NEAT1 or SC35 in 17-19 regions of interest (ROIs). Central lines in boxes define medians, top and bottom limits define upper and lower quartiles, respectively. Whiskers show the full data range, except for outliers. Grey dots are individual data points. Significance of positive or negative correlations was assessed using the Costes method of random ROI shifts. Highly significant (p<0.01) positive correlations between vDNA and CPSF6 intensities are found in 16 out of 19 ROIs; highly significant negative correlations between vDNA and NEAT1 are found in 19/19 ROIs, and highly significant positive correlations between vDNA and SC35 are found for 16/17 ROIs. Data are from one experiment. For CPSF6, the experiment was repeated with similar results. (E) Intensity profiles of EdU and DAPI along the dotted lines in the vDNA images above.
Figure 5CD69 expression, an early marker of lymphocyte activation, in CD3+ T cells infected with shNC, shNF45 or shNF90 lentivirus upon stimulation with P/I for 12 h. B-C PBMC cells with shNC, shNF45 or shNF90 lentivirus infection were stimulated with P/I for 12h, then the freshly replaced medium without P/I after another 24 h culture was harvested for IL-2 and IFN-γ detections by ELISA.(n=3) The proliferation of shNC, shNF45 or shNF90-infected PBMCs was monitored using a CCK-8 assay. (n = 3) Confocal images to show the subcellular localization of NFATc2 in CD3+ T cells treated with P/I and FK506 or CX5461. Scale bar, 10 μm. Flow cytometry analysis of CD69 expression in P/I-stimulated CD3+ T cells treated with or without FK506 or CX5461. G-H ELISA assays of IL-2 and IFN-γ in CD3+ T cells with the indicated treatments. (n = 3) Flow cytometry analysis of CFSE-labeled CD3+ T cells in the presence of DMSO, FK506 and CX5461 for 0, 3 and 7 d.
Endogenous MIR376A limits starvation-induced autophagy.Figure 7(A) Blockage of endogenous MIR376A by Ant-376a, but not CNT-Ant further stimulated starvation (STV)-activated LC3-I to LC3-II conversion in MCF-7 cells. ACTB was used as a loading control. LC3-II/LC3-I densitometric ratios were marked. (B) Ant-376a, but not CNT-Ant resulted in further activation of SQSTM1 protein degradation following starvation in MCF-7 cells. SQSTM1/ACTB ratios were marked.
Figure 2(A) Control 143B cells (left panels), COX1∆ cybrids (middle panels), and COX2∆ cybrids (right panels). The right lane highlights structural subunits of CI (yellow), CIII (light green), and CIV (orange brown), and assembly factors (orange). Intensity-based absolute quantification (iBAQ) values were normalized to the sum of all control values. Top lanes indicate gel slice numbers and their relative native masses. (B) Intensity profiles showing abundance and relative distribution of structural subunits from CI (upper graphs), CIII (middle graphs), and CIV (bottom graphs). The identities of MRC complexes and SCs are as in Figure 1.
Figure 5SSA/RMD and BIR/SSA are both dependent on ATM. SSA/RMD and BIR/SSA frequencies in U2OS (EGFP-SSA-3427-17) cells were determined after the addition of ATM inhibitor (20mM ku55933) or DMSO to the cell culture medium 24 hours before transfection of the indicated sgRNA/Cas9 BIR/SSA are both dependent on ATM. BIR/RMD in mES cells carrying the RMD-GFP reporter were determined after the addition of ATM inhibitor (20mM ku55933) or DMSO to the cell culture medium 24 hours before transfection of the indicated sgRNA/Cas9 or in ATM KO mES cells carrying the RMD-GFP reporter (right). Dependence of RMD on H2AX. SSA/RMD and BIR/RMD frequencies in U2OS (EGFP-SSA-3427-17) cells were determined using the indicated sgRNA/Cas9 after depletion of H2AX by lentivirus expressed shRNA Depletion of H2AX by shRNA in was determined by qPCR D, Dependence of RMD on H2AX. BIR/RMD in mES cells carrying the RMD-GFP reporter , or by transfection with H2AX siRNA or in RAD52 KO cells
Figure 3(A) Euc1 domain structure is reminiscent of transcription factors. Top: Schematic representation of Euc1, with a predicted coiled-coil domain (CC) and GCR1-domain indicated. Predicted DNA binding of the C-terminal part was confirmed in Y1H experiments (Appendix Fig. S3A-C). Bottom: Euc1-like protein sequences from closely related Saccharomyces species were aligned and a phylogenetic tree was generated using Clustal Omega. Jalview was used to graphically display the degree of sequence conservation (see also Appendix Fig. S4A). (B) The ub-HS-motif is conserved in closely related yeast species. The 7 yeast Multiz Alignment & Conservation tool of the UCSC Genome Browser was used to retrieve alignments of sequences corresponding to ub-HS-motifs from other Saccharomyces species. Dot (.) indicates a conserved base. (C) Euc1 can induce transactivation via its N-terminal 30 amino acids. Euc1 constructs under the endogenous EUC1 promoter were transformed in a reporter strain as described for Fig. 2D and serial dilutions were spotted on control or selective media to test HIS3-activation. Cells were grown at 30°C for 3 days. (D) Quantification of HIS3 mRNA levels from strains used in (C). Cells were grown in liquid media with selection for the transformed plasmids (SC-Leu), harvested in logarithmic growth phase and total mRNA was prepared. After reverse transcription, HIS3 mRNA levels were quantified using qPCR (RT-qPCR), normalized first to ACT1 mRNA and then to the empty vector control strain. Data represent means ± SD (n = 4). p = 2.43*10-5 (Student's t-test).
Arginine in hnRNPA2 LC is required for the interaction with granule component hnRNPF PLD.Figure 1A) hnRNPA2 LC (AlexaFluor488-tagged, green) undergoes LLPS, while hnRNPF PLD (AlexaFluor555-tagged, red) does not. However, hnRNPF PLD partitions into hnRNPA2 LC droplets when mixed at a 1:1 ratio. Conditions: 20 µM indicated protein (~1% fluorescently tagged), 20 mM MES pH 5.5, 50 mM NaCl, 150 mM urea. Scale bar: 10 µm. B) At 300 µM, FUS LC (AlexaFluor488-tagged, green) undergoes LLPS, but at 200 µM hnRNPF PLD (AlexaFluor555-tagged, red) still does not undergo LLPS. When mixed at 300 µM FUS LC and 200 µM hnRNPF PLD, hnRNPF PLD does not partition into FUS LC droplets. Conditions: 300 µM FUS and 200 µM hnRNPF PLD (~1% fluorescently tagged), 20 mM MES pH 5.5, 150 mM NaCl, 150 mM urea. Scale bar: 10 µm. C) While FUS LC and hnRNPF PLD both have a small negative predicted net charge at neutral pH, hnRNPA2 LC has a predicted +4 net positive charge, due to the 9 positively charged residues (8 arginine, 1 lysine) and 5 negatively charged residues. D) Removal of the charged residues from hnRNPA2 LC (hnRNPA2 LCCD) prevents partitioning of hnRNPF PLD into the hnRNPA2 LC phase. Addition of hnRNPA2 LC-like charged residue patterning to FUS LC (FUS LCCE) allows the partitioning of hnRNPF PLD at 40 µM. Conditions: protein concentration indicated next to image (20 µM hnRNPA2 LC and hnRNPA2 LCCD, 40 µM FUS LCCE, hnRNPF PLD concentration matches other protein in mixture (either 20 or 40 µM)) (all ~1% fluorescently tagged), 20 mM MES pH 5.5 50 mM NaCl, 150 mM urea. Scale bar: 10 µm. E) Substitution of all arginines in hnRNPA2 LC with lysine prevents the partitioning of hnRNPF PLD into hnRNPA2 LCRK droplets. Removing all charged residues except for arginine from hnRNPA2 LC (hnRNPA2 LCCD,R) allows partitioning of hnRNPF PLD into droplets, indicating arginine in hnRNPA2 LC is required and necessary for hnRNPF partitioning. hnRNPA2 LCRK does not phase separate much as hnRNPA2 LC at these conditions, see Figure S1J for quantification of phase separation of variants. Conditions: 20 µM proteins, 20 mM MES pH 5.5 50 mM NaCl, 150 mM urea. Scale bar: 10 µm. F) Quantification of phase separation of hnRNPA2 LC constructs used to determine the residue types important for hnRNPF PLD partitioning. hnRNPA2 LCNS (purple) has similar phase separation to hnRNPA2 LC. hnRNPA2 LCCD (red) is consistently phase separated with ~5 µM protein remaining in the supernatant at all salt conditions tested. Adding back arginines to hnRNPA2 LC no charge (hnRNPA2 LCCD,R, green) brings phase separation as a function of salt to similar levels as hnRNPA2 LC. Changing all the arginine residues to lysine (removing the π-character but maintaining positive charge, hnRNPA2 LCRK) also removes the salt dependence of phase separation but has reduced phase separation overall. Conditions: 20 µM of each protein, pH 5.5 MES, NaCl concentration as indicated, 25° C. Error bars are standard deviation of three replicates.
Figure 4A. Amino acids of exon A. Positive charged amino acids are marked blue, negative charged ones are shown in red. Mutated regions are in bold and different mutant combinations investigated in Fig. EV5 are depicted below. B. Traces showing average changes (mean±s.e.m.) in intracellular Ca2+ (Fura2 ratio) over time in response to perfusion of different [Ca2+]o as indicated in the upper bar with constructs as indicated expressed in HEKO1. STIM1 (black, n=145), STIM1A (red, n=157) and STIM1A_D503A (n=185). C. Relative fluorescence intensities of mCherry-tagged constructs measured in (B). D. Quantification of changes in resting ratio, TG peak (∆ ratio) and SOCE parameters measured in B. E. Data information: *P<0.05, **P<0.01 ***P<0.001 Kruskal-Wallis Anova with Dunn's multiple comparisons test. Data points (total n) were obtained from three biological replicates with each three technical replicates (each multiple cells) and shown as mean±s.e.m for traces and as scatter plots with the underlying boxes showing the means for individual parameters. E. Average traces showing whole-cell current density (CD) over time extracted at -80 mV in HEKO1 cells transfected with STIM1 (black), STIM1A (red) or STIM1A_D503A (blue). F. Average maximum CDs recorded from cells measured in E (n within bars). G Data information: *P<0.05, **P<0.01 ***P<0.001 Kruskal-Wallis Anova with Dunn's multiple comparisons test. Data points (total n) were obtained from three biological replicates with each three technical replicates (each multiple cells) and shown as mean±s.e.m for traces and as scatter plots with the underlying boxes showing the means for individual parameters. G. Average current-voltage (I- V) relationship of all cells recorded in E.
Figure 8EUC1 displays negative genetic interactions with genes involved in general and specific transcriptional regulation (A) and in particular with members of the Rpd3L histone deacetylase complex (B). For (A-D), serial dilutions of the indicated strains were spotted and grown on YPD control or selective media plates, or under conditions as indicated. Note that all strains used in (A-D) contained an extra copy of MED11, EUC1 displays negative genetic interactions with genes involved in particular with members of the Rpd3L histone deacetylase complex For , serial dilutions of the indicated strains were spotted and grown on YPD control or selective media plates, or under conditions as indicated. Note that all strains used in (A-D) contained an extra copy of MED11, For serial dilutions of the indicated strains were spotted and grown on YPD control or selective media plates, or under conditions as indicated. Note that all strains used contained an extra copy of MED11 (C) Plasmid-borne EUC1 complements genetic interactions with Rpd3L subunits. Empty vector (-) or a plasmid encoding EUC1 with its endogenous promoter were transformed in the indicated strains and spotted on selective media. For serial dilutions of the indicated strains were spotted and grown on YPD control or selective media plates, or under conditions as indicated. Note that all strains used contained an extra copy of MED11 (D) EUC1 and Rpd3S act in a common pathway which is redundant with Rpd3L. rco1∆ and eaf3∆ (Rpd3S) show similar phenotypes when paired with sds3∆ (Rpd3L) as euc1∆ and are epistatic with euc1∆. (E) Graphic summary of EUC1 genetic interactions as tested in (A-D) and Appendix Fig. S10. Arrows indicate negative genetic interactions upon cold (blue), heat (red) or DMSO stress (black), dashed grey arrows indicate epistatic relationships.
Figure 3A) The table shows putative miR-424(322)/503 targets selected from the TargetScan database and ranked based on their probability of conserved targeting. A schematic representation of the canonical Wnt pathway with arrows indicating the putative targets that were investigated is also shown. B) The upper panel illustrates the location of the conserved and non-conserved (-nc) predicted microRNA binding sites in the 3'UTR of 3 selected target genes. The bar graph compares the normalized expression of a firefly luciferase reporter (luc) plasmid (EV) after transfection in 293T cells and the same luc reporter with a modified 3'UTR containing predicted microRNA binding sites. The values represent the ratio of luc expression when the reporter was co-transfected with synthetic miR-424 and miR-503 vs. a control non-targeting sequence. The bars with a diagonal line pattern indicate non-conserved sites. A known miR-424/503 binding site located in the 3'UTR of CDC25 is used as a positive control. The horizontal line indicates 25% of reduction in luminescence and it is included as a visual reference. The error bars show the standard deviation. C) Luciferase assays showing that the effect of the miR-424 and miR-503 on luc expression is abrogated when the seed sequence in the microRNA binding sites was mutated. The study was performed in four sites statistically significant from panel B. The error bars show the standard deviation. D) Western blot of LRP6 and TBLR1 in the miR-cluster-Dox-MCF-10A model upon upregulation of miR-cluster with 100 ng/ml of doxycycline for the specified number of days. The numbers below the blots indicate the quantitation by densitometry of protein expression relative to β-actin. The expression level of miR-424 and miR-503 is indicated in the bar graph below the blots. UN indicates samples untreated with doxycycline. The error bars show the standard deviation. E) Western-blots of LRP6 and TBLR1 in parental MCF-10A cells after transfection with miRNA hairpin inhibitors against miR-424/503. F) Western blot of LRP6 and TBLR1 expression in WT and miR-424/503-KO basal cell-derived organoids after 14 days in culture with and without Wnt3a (100ng/ml). The arrow shows the correct TBLR1 band. G) IHC staining showing that LRP6 accumulates in the epithelium of female miR-424(322)/503-/- mice (1-year-old). The scale bar indicates 100 micrometers (µM).
Figure 3A: Live cell imaging results showing in vitro meiotic division of WT and Cnot6l-/- oocytes. Abbreviations: PM, prometaphase; MI, metaphase I; AI, anaphase I; TI, anaphase I-metaphase II transition; MII, metaphase II; PB1: polar body-1. Hours after released from GV arrest were indicated. Scale bar, 20 μm B: Data represent the mean and standard deviations of mCherry-Securin fluorescence intensity in WT and Cnot6l-/- oocytes at each time point. Values from individual oocytes are normalized relative to that at 0 h after released from GV arrest. The numbers of analyzed oocytes were indicated (n) C: SMC3 immunofluorescence showing cohesin on chromosomes of WT and Cnot6l-/- oocytes at 16 h after culture. Centromeres and DNA were labeled by CREST and DAPI, respectively. Scale bar, 5 μm D: BUB3 immunofluorescence on chromosome spreads made from WT and Cnot6l-/- oocytes at 9.5 h after culture. More than 8 oocytes were observed for each genotype with similar results. Scale bar, 5 μm E: PB1 emission rates in WT and Cnot6l-/- oocytes cultured with or without MPS1 inhibitor reversin (5 μm). Reversin is added at 9 hours after culture. Error bars, s.e.m. The numbers of analyzed oocytes were indicated (n)
LDM chemotherapy offsets HIF-1α levels in breast cancer metastases to the lung A: EMT6-CDDP lung metastatic nodule size.Figure 3A: EMT6-CDDP lung metastatic nodule size. Left: Histogram of cross-sectional metastatic diameter (Ø). Middle: Metastatic diameter (Ø) by chemotherapy regimen. F(5,99) = 0.9310 and P= 0.4644 (not significant) for overall treatment by Brown-Forsythe ANOVA. Right: Dichotomized metastatic size at median diameter of vehicle-treated tumors. Lung nodule size was classified as small (blue) or large (red). Individual nodule counts per group and size category (small/large) are plotted on the right ordinate. χ2 (df=5) = 2.631; P= 0.7567 (not significant) for overall effects on size. B: Hypoxic fraction in lung metastatic nodules. Pimonidazole adduct immunoreactivity was automatically quantified and expressed as fractional positive areas per nodule (rightmost panel). F(5,110) = 7.544 and P< 0.0001 for overall treatment by Brown-Forsythe ANOVA; *, P< 0.01 vs vehicle; #, P= 0.0111 doublet LDM cyclophosphamide + capecitabine vs LDM cyclophosphamide + MTD capecitabine by Benjamini, Krieger and Yekuteli post hoc test. C: HIF-1α levels in lung metastatic nodules. Automatic quantification of HIF-1α levels measured as fractional positive areas per nodule (rightmost panel). F(5,114) = 5.325 and P= 0.0004 for overall treatment by Brown-Forsythe ANOVA; *, P< 0.01 vs vehicleby Benjamini, Krieger and Yekuteli post hoc test. D: Microvessel density in lung metastatic nodules. Automatic quantification of CD31 fractional areas per nodule (rightmost panel). F(5,124) = 7.531 and P< 0.0001 for overall treatment by Brown-Forsythe ANOVA; *, P< 0.01 (LDM capecitabine, cyclophosphamide, or doublet capecitabine + cyclophosphamide vs vehicle), #, P= 0.0417 (doublet LDM capecitabine + cyclophosphamide vs doublet LDM cyclophosphamide + MTD capecitabine) by Benjamini, Krieger and Yekuteli post hoc test.
Figure 1A The flowchart depicting the transcriptomic profiling of isolated worm tissues. Isolated cells from each tissue were pooled for Smart-seq2. B, C Principal component analysis (B) and hierarchical clustering (C) of the transcriptomic datasets of different tissues from young and aged worms in three biological replicates. D1: day 1 of adulthood, D8: day 8 of adulthood. Neu: neuron, Int: intestine, BWM: body wall muscle, Hyp: hypodermis, Coel: coelomocyte. D The volcano plots showing the differentially expressed genes in ageing tissues. Genes with a fold change greater than 1.5 and a P value smaller than 0.05 were considered significantly changed by ageing. The numbers and percentages of Age-DEGs in expressed genes are shown in the corresponding plots.
Flowchart of exome sequencing filtering outcomes.Figure 1Whole exome sequencing was initially performed on DNA extracted from peripheral blood leukocytes of 111 individuals from the 18 most extensive families from our cohort (76 with DP and 35 controls). The exome sequences were aligned to the UCSC hg19 reference genome. Picard tools and the genome analysis toolkit were used to mark PCR duplicates, realign around indels, recalibrate quality scores and call variants. Variants were analyzed further and filtered for potential causal variants using filters for quality control, predicted functional annotation, minor allele frequency (MAF), segregation with trait, variants in multiple families and biological relevance (See methods and Table EV1 for further information on filtering criteria). Targeted exome sequencing using a Fluidigm array of 28 candidate genes identified post-filtering was then performed in a further 42 families from the same cohort (288 individuals, 178 with DP and 110 controls). Variants post targeted re-sequencing were filtered using the same criteria as the whole exome sequencing data. Rare variant burden testing was performed for all genes selected for targeted re-sequencing, in order to rank candidate genes post targeted re-sequencing. A multiple comparison adjustment was applied to the set of 28 p values post hoc (Benjamini et al., 2001). Screening of 100 further cohort controls was via conventional Sanger sequencing. Functional annotation of the variants as described elsewhere in methods. DP - delayed puberty. *data unpublished.
Figure 7A. Time course of translocation (Nuclear NFAT-GFP signal/cytosolic signal normalized to TG addition at t=0, mean±s.e.m, for each time point) after transfection of indicated constructs and preincubation with 1 µM PF-04957325 (analyzed cells: STIM1 + PF n = 75, STIM1A + PF n = 50, ØSTIM + PF n = 9) Data (total n as indicated) from at least three independent transfections. * p< 0.05, **p<0.01, ***p<0.001, Kruskal-Wallis Anova with Dunn's multiple comparisons test. B. PF-04957325 induced difference of the nuclear NFAT-GFP signal vs. cytosolic signal from (A) to mean of control without the blocker (see Fig. 6D). * p< 0.05, **p<0.01, ***p<0.001,Mann-Whitney test of differences.. C Data information: *p<0.05, **p<0.01, ***p<0.001, 2way Anova Data (total number of cells, n) were obtained from at least three independent transfections with several technical replicates each. C. Endpoint value with addition of PF or indicated cAMP analoga loaded for 30 min as AM esters before the experiment, shown as mean±s.e.m , ØSTIM + 6Bnz-cAMP/8-pCPT-cAMP n = 15/14 cells; STIM1 + 6Bnz-cAMP/8-pCPT-cAMP: n = 87/66, STIM1A + 6Bnz-cAMP : n = 78/67; from three independent transfections. Data information: *p<0.05, **p<0.01, ***p<0.001, , Kruskal-Wallis Anova with Dunn's multiple comparison test Data (total number of cells, n) were obtained from at least three independent transfections with several technical replicates each. D. Traces showing average changes (mean) in intracellular Ca2+ (Fura2 ratio) over time in response to perfusion of TG as indicated in the upper bar in SH-SY5Y S1-/- cells transfected with STIM1,STIMA or STIM1A_D503A IRES mCherry and measured with and without incubation with 1 µM PF-04957325 (n=82-134). E. Area under the curve calculated from individual cells as measured in (D). Bars indicate mean±s.e.m. Data information: *p<0.05, **p<0.01, ***p<0.001 , Kruskal-Wallis Anova with Dunn's multiple comparison test Data (total number of cells, n) were obtained from at least three independent transfections with several technical replicates each. F. schematic representation of the RflincA, modified after (Ohta et al., 2018). G. Normalized fluorescence signal change (mean±s.e.m) of R-FlincA, SH-SY5Y S1-/- cells expressing STIM1 (n=20) or STIM1A (n=22) IRES GFP variants and the indicator were stimulated by TG (1 μM). To avoid photobleaching, values were only recorded after 5 and 30 min, respectively The mutant R-Flinc (n=15) was measured in SH-SY5Y wt cells after TG stimulation. Data information: *p<0.05, **p<0.01, ***p<0.001, Kruskal-Wallis Anova with Dunn's multiple comparison test Data (total number of cells, n) were obtained from at least three independent transfections with several technical replicates each.