EMBO/sd-character-level-ner huggingface dataset:
Figure 6(A) Schematic representation of the m220 stromal feeders expansion culture to compare the meiotic capacity of XGFP- and XGFP+ PGCLCs. Meiosis is induced via addition of Retinoic Acid (RA) and Bone Morphogenetic Protein 2 (BMP2). c0 = starting day of culture, c9 = culture day 9 and last day of culture. (B) Representative images for the expression of XGFP (green) and SYCP3 (red) in germ cells at c5, c7 and c9 from XGFP+ and XGFP- PGCLCs. All cells with SYCP3 signal irrespective of localization pattern or intensity were scored as SYCP3+ cells. Cells were counterstained with DAPI (grey). Scale bars = 10 μm. (C) Number of SYCP3+ cells per m220 culture day originating from XGFP+ and XGFP- PGCLCs. Each white dot represents a biological replicate (n = 3). P-values shown are from a two-sided Mann-Whitney U test. n.s. = not significant. (D) Percentage of XGFP+ cells among SYCP3+ cells at the indicated m220 culture day, originating from XGFP+ or XGFP- PGCLCs. Green bars show constitutive GFP expression in XGFP+ PGCLC-derived cells, while striped bars signify XGFP reactivation in XGFP- cells. Each white dot represents a biological replicate (n = 3). (E) Representative images showing stages of meiotic prophase I from culture day 9 (c9) germ cells from XGFP+ and XGFP- PGCLCs. c9 germ cells were spread and immunostained for SYCP3 (red), and γH2AX (grey). Scale bars = 10 µm. (F) Quantification of meiotic progression in culture day (c9) expanded germ cells derived from XGFP+ and XGFP- PGCLCs. The graphs show the percentages of the meiotic stage for SYCP3+ cells. Lepto, leptotene; Zygo, zygotene; Pachy, pachytene; Diplo, diplotene. Numbers indicate detected cells per meiotic stage and original XGFP status. (G) Representative tile scan merged images of XGFP (green) and Xtomato (red) in germ cells at c7 from XGFP+ and XGFP- PGCLCs. Cells were counterstained with DAPI (grey). Scale Bar = 1 mm. The white squares contain a magnified image of the region depicted by the white dotted squares (Scale Bar = 1 µm). (H) Density ridge plot showing size of cellular aggregates in (G) measured using the XTomato signal. X-Axis shows area in µm2 scaled as log2. Dashed line represents the mean. XFGP- mean = 1320 µm2, XGFP+ mean = 5514 µm2. XGFP- n = 297 aggregates, XGFP+ n = 373 aggregates. P-value from two-sample unpaired Wilcoxon-Mann-Whitney test with R defaults.
Wnt/Ca2+ pathway modulates vesicle transfer in primary neurons.Figure 6Top: Schematic of the culture systems used to assess cell-to-cell contact-dependent or secretion-mediated transfer of vesicles in primary neurons. After dissection, neurons were labeled in suspension with DiI (donor, D) or CTG (acceptor, A). In the co-culture system, neurons were plated in 1:1 ratio and cultured for 20 h and then treated with 200 ng/mL of Wnt5a or Wnt7a for 4 h. In the secretion-based system, donor neurons cultured alone for 20 h were treated with 200 ng/mL of Wnt5a or Wnt7a for 4 h, and the medium was then collected and replaced for the medium of acceptor neurons cultured separately. Acceptor neurons were further incubated with the conditioned media of donor neuron for 24 h. Bottom: Representative confocal images of co-cultured donor and acceptor neurons. The insets (right panels) show a magnification of the area depicted in the expanded field images (left). Yellow arrows point to DiI-labeled vesicles inside acceptor neurons. Percentage of acceptor neurons containing DiI-labeled vesicles after being co-cultured with donor neurons and incubated for 4 h with the indicated treatments. Percentage of acceptor neurons containing DiI-labeled vesicles after being cultured for 4 h with the conditioned media of treated donor neurons. Confocal image showing a donor (D) and an acceptor (A) neuron connected by TNT-like structures (yellow arrowhead in the merged panel). Neurons were stained with WGA (white) and DAPI (blue). The insets (right panels) show a magnified image of the area depicted in the expanded field (left).
Figure 2 A The irc5-Δ1 mutant exhibits S-phase delay in the presence of MMS. DNA content of wild type (W303-1a) and irc5-Δ1 (IL012) cells was measured by FACS and plotted as histograms  B Completion of DNA replication is delayed in the irc5-Δ1 mutant after MMS treatment. Intact chromosomes were isolated from wild type (W303-1a) and irc5-Δ1 (IL012) cells and processed for PFGE. M, 0.03% MMS  C Quantification of data presented in B. Error bars represent mean value ± standard deviations of mean for the intensity values of three distinct chromosome bands representing chromosome XII or III (n=3). Values for G1 were set to 100  D Samples used for PFGE in B were processed for FACS analysis
Endothelial Ang-2 expression reduced pericyte coverage and increased macrophage infiltration in experimental glioblastoma.Figure 2Figure 2: Endothelial Ang-2 expression reduced pericyte coverage and increased macrophage infiltration in experimental glioblastoma. GL261 cells were intracerebrally transplanted in wild type or Ang-2 gain-of-function mice (Ang-2 DT). Brain tumor sections were stained with antibodies against CD31 and desmin (A), and microvessel densities (MVD) and pericyte coverage were determined (WT n=14; Ang-2 DT n=6) (B). FACS analysis of infiltrating macrophages in Ang-2 DT normal brain (w/o tumor) compared to WT is shown (C, dot plot; D, quantification) (WT n=5; Ang-2 DT n=3). ). Immunohistochemistry staining for monocytes/macrophages (F4/80) and neutrophils (Ly6G; arrowheads) in brain tumors derived from WT or Ang-2 overexpressing mice (E), Quantitative analysis of C (F) (WT n=14; Ang-2 DT n=10) Kaplan-Meier survival curve of GL261 tumor-bearing WT and Ang-2 DT mice (WT n=17; Ang-2 DT n=14) (E). Statistical analyses were performed using an unpaired Student´s t-test (B, F), one-way Anova (Tukey post test) (D), Log-rank and Wilcoxon (G) * p < 0.05, ** p < 0.01; data are mean ± SEM
Figure 7A Largely mutually exclusive localization of LC3 and K48 chains. GFP-LC3 expressing HeLa cells were fixed 2 h after LLOMe washout and stained for endogenous LAMP1 and K48 or K63 chains. Correlation coefficients were calculated for colocalization of LC3 with K63 or K48. The whiskers represent 10th and 90th percentile. Points below and above the whiskers are drawn as individual dots. The single line indicates the median. *** = p < 0.001 (Mann-Whitney-U test). B Lipofectamine-coated latex beads damage lysosomes and induce their K48-ubiquitination. Cells expressing mCherry-Gal3 were incubated with Lipofectamine-coated latex beads for 3 h and stained with K48 chain-specific antibodies. C YOD1 association and dissociation precedes LC3 recruitment. Live cell video microscopy stills of cells co-expressing mCherry-YOD1-wt and GFP-LC3 after treatment with Lipofectamine-coated latex beads for 30 min at 2 min image intervals. D Stabilization of K48-conjugates on lysosomes impairs recruitment of LC3. Cells expressing GFP-YOD1-CS were treated as in (A) and stained for LAMP1 and p62 or LC3. Percentage of GFP-YOD1 vesicles positive for p62 or LC3 was determined (mean ± SD). E Depletion of ELDR components impairs LC3 recruitment for autophagosome formation. Cells were transfected with control (Ctrl), p97 or YOD1 siRNA oligos, treated with LLOMe, fixed after 12 h chase and stained for endogenous LC3 and K48 chains. Quantification of the number of K48-positive vesicles per cell and number of LC3-positive K48 vesicles per cell was determined. Data represent mean ± SD of three independent experiments. Scale bars, 10 µm.
Figure 4(A) Survival curves of Ctrl (Npm1F/FVav1Cre-), cKO (Npm1F/FVav1Cre+), p53 KO (Npm1F/Fp53-/-Vav1Cre-) and DKO (Npm1F/Fp53-/-Vav1Cre+) mice. Overall survival of the indicated mice was examined by plotting Kaplan-Meier survival curves. Log-rank test was used to generate p values, *** p<0.001. (B) Amount of total bone marrow mononuclear cells (BM-MNCs) in Ctrl, 4-months old Npm1 cKO mice and moribund Npm1 cKO (cKO†) (n = 3-5 mice per group). (C) Npm1 and p53 protein levels in KSL cells of Ctrl (Npm1F/FVav1Cre-), and cKO (Npm1F/FVav1Cre+) mice. (D) p21 expression levels in BM-MNCs of Ctrl and Npm1 cKO mice (4 months old, n = 4 mice per group). (E) White blood cell (WBC) count (left) and myeloid fraction (CD11b+Gr-1+, right) in PB (n = 3-8 mice per group). (F) Representative PB smears of DKO mice. Insets show anisocytosis (a), poikilocytosis (b) and dysplastic myeloid cell (c). DKO mice show dysplastic erythroid cells (polychromatophilic, blue arrowhead), Howell-Jolly bodies, (black arrowhead in insert a), dysplastic monocytes (black arrow, and i-v), dysplastic neutrophils (vi), a pseudo-Pelger-Huet anomaly (ii), and dysplastic platelets (giant platelet, iv and vii). Scale bars, 10μm. (G) Pictures of spleens (top) and spleen weight quantification (bottom, n = 3-5). (H) Percentages of myeloid fraction (CD11b+Gr1+), c-Kit positivity, and B220 positivity in spleen samples (n = 3-5). (I) Representative H&E staining of sections of the spleen of 2-month-old DKO mice with myeloid disorders. Arrowheads (right) indicate the infiltrating myeloid blasts. Scale bars, 250 μm (left) and 10μm (right), respectively. (J) Myeloid subtype (CD11b+Gr1mid), and c-Kit positivity inside myeloid fraction in BM (n = 3-5). (K) c-Kit+Sca1-Lin- (KL Sca1-) and granulocyte-macrophage progenitor (GMP) in the BM (n = 3-4).
Study design, datatypes & CMS prediction for cell lines.Figure 1(A) Overview of the multi-omics data integration workflow followed in this study. Proteomic and transcriptomic data generated as part of this study or available from the literature for colorectal cancer (CRC) cell lines and patients were integrated in order to identify cell lines and tumours forming proteomic subtypes. Four published drug sensitivity datasets (abbreviated CCLE, CTRP, GDSC and Cetuximab (Medico et al.); one dose response plot for each data source in shown) were overlaid onto the proteomic data to identify protein signatures associated with sensitivity or resistance. An example of an effect-size heat map for one drug, ten proteins and 20 cell lines is shown at the bottom-right (see main text and Appendix for details). (B) Circos plot visualising the different datasets that were integrated in this study. The dendrogram in the centre hierarchically ordered tumours (violet 'Dataset' label) and cell lines (blue 'Dataset' label) based on the mRNA expression of classifier genes from Guinney et al. (2015). A set of rings around the dendrogram indicates which proteomics data (Full proteome, Kinome), mRNA technologies (Agilent microarray, Genome-Analyser-based mRNA-Seq, HiSeq-2000-based mRNA-Seq, Affymetrix microarrays or Illumina Beadarrays) and drug sensitivity datasets (Cetuximab, CCLE, CTRP or GDSC) were included in this study. The outermost ring indicates the membership of cell lines/tumours in a consensus molecular subtype ('CMS'). Undetermined CMS class labels or unavailable data were left white (see main text and Appendix for details). See also Figure EV1.
Figure 3A. Western blot analysis of indicated vsv-MAD1-WT or 3EK HeLa clones treated with or without doxycycline for 10 days. B. Immunofluorescence images showing MAD1 and ZW10 kinetochore levels in nocodazole-arrested MAD1-WT-C13 AND 3EK-C14 just after nuclear envelope breakdown (early prometaphase) or later in mitosis when the chromatin is condensed (late prometaphase). Note, that early and late prometaphase was defined based on the level of chromatin condensation. C,D. Relative kinetochore volumes occupied by MAD1 (C) and ZW10 (D) (relative to CenpC) in MAD1-WT and -3EK cells in early and late prometaphase. E. Quantification of MAD1 kinetochore intensity from indicated MAD1-WT and 3EK clones treated as in (B).
Stat3 deletion in astrocytes protects from spatial memory and learning deterioration (A) Spatial learning and memory were assessed in the Morris Water Maze paradigm.Figure 7Spatial learning and memory were assessed in the Morris Water Maze paradigm. APP/PS1-Stat3KO mice showed faster latencies to reach the hidden platform compared with APP/PS1-Stat3WT on days 4 and 5, but were similar to WT-Stat3WT and WT-Stat3KO mice (Two-way repeated-measures ANOVA followed by Bonferroni post-hoc test; P values are for APP/PS1-Stat3KO vs. APP/PS1-Stat3WT mice). The area under the curve (AUC) for the latency to reach the hidden platform was similar in APP/PS1-Stat3WT compared to WT-Stat3WT and WT-Stat3KO mice, but significantly higher in APP/PS1-Stat3KO mice (Kruskal-Wallis test followed by Dunn's multiple comparisons test). The swimming velocity was similar in all groups (Kruskal-Wallis test followed by Dunn's multiple comparisons test). In the probe trial 24 h after the last training day, the time mice spent in the target quadrant (TQ) was different from chance in all groups except for APP/PS1-Stat3WT mice (p < 0.05, one-tailed one-sample t test). APP/PS1-Stat3KO, WT-Stat3WT and WT-Stat3KO mice spent significantly more time in the target quadrant compared to the mean of all other quadrants (AO), whereas APP/PS1-Stat3WT spent equal times in the target and all other quadrants (Wilcoxon matched-pairs signed rank test for each comparison; APP/PS1-Stat3WT, n = 15 (8 female and 7 male) mice; APP/PS1-Stat3KO, n = 15 (9 female and 6 male) mice; age, 8-9 months).
Figure 3A. Confocal time-lapse live imaging was performed in Tg(c-myb:GFP;kdrl:mKate-CAAX) embryos from 28 to 42 hpf (Movie EV 1). Shown are three representative time-points in which the transformation from hemogenic endothelial cells to the hematopoietic fate is visible, indicated by the white arrowheads. For each time-point merged images are shown. White arrowheads denote double-positive cells.B. Cell counts of total numbers of kdrl+cmyb1+ cells "in view", dividing and respectively emerging HSPCs. n=10 movies from n=3 biological replicates were analyzed. A non-parametric Mann-Whitney test was used to test for statistical significance and error bars are shown as ± s.d. C. Gene expression analysis of endothelial (kdrl, flt1, dab2, cdh5) and blood specific genes (c-myb, CD45, CD41, runx1) in sorted kdrl+c-myb+ cells from 34 hpf Tg(c-myb:GFP;kdrl:mKate-CAAX) embryos. Cells were isolated from 15-25 pooled embryos per sample. Three biological experiments were performed with one representative shown. Error bars indicate s.d. of three technical replicates for each representative experiment.
The three different migration modes.Figure 1A-C: Cell outlines at different times for the three distinct cell migration modes: fan-shaped cells (A), oscillatory cells (B), and amoeboid cells (C) (scale bar: 10μm).
Figure 5Intraperitoneal GTT after 19 weeks of HFD-feeding, glucose injections at 1.5 mg per g of body weight (n = 11-13 animals per group). Area under the curve calculated from (A) (n = 11-13 animals per group). Plasma insulin levels during GTT (n = 11-13 animals per group). Intraperitoneal ITT after 20 weeks of high-fat diet intervention. Insulin was adjusted to 1 mU per g of body weight (n = 11-13 animals per group). Area under the curve determined from (D) (n = 11-13 animals per group). Plasma insulin levels after a 5-hour fast, before injecting insulin (n = 11-13 animals per group).
ROCK inhibition promotes the collective invasion of colorectal adenocarcinoma explants A) Representative images of organoids formed from patient derived xenografts (PDXs) that have been embedded in collagen-I gels during 3 days, in non-treated (NT) or ROCK inhibitors treated-conditions (+Y27632) and (+H1152).Figure 4A) Representative images of organoids formed from patient derived xenografts (PDXs) that have been embedded in collagen-I gels during 3 days, in non-treated (NT) or ROCK inhibitors treated-conditions (+Y27632) and (+H1152). At end-point, organoids were stained for E-cadherin, F-actin (Phalloidin) and nuclei (DAPI). i and ii are high magnification of boxed regions of +Y27632 and +H1152 organoids respectively. Arrows point to protruding cells. White stars show nuclei that engage in protrusions. Scale bars: 20μm. B) The bar graph represents the percentage of protrusive organoids resulting from three independent experiments. Over 50 organoids were counted per condition (Means ± SEM). P values were calculated using paired t-test (***p<0.001). C) Representative images of human adenocarcinoma explants after 2 days in 3D collagen-I gel, in non-treated (NT) or ROCK inhibitors treated-conditions (+Y27632) and (+H1152) and stained for the apical pole (Ezrin), F-actin (Phalloidin) and nuclei (DAPI). Boxed regions of NT, +Y27632, and +H1152 explants are shown at high magnification. Arrowheads point to non-protruding cells. Arrows point to protruding cells. White stars show nuclei that engage in protrusions. Scale bars: 20μm. D Explants displaying (D) lumens from 10 patients were quantified and results presented as percentage of total explants. Over 50 explants were counted per condition (duplicate) and per patient(Means ± SEM). P values were calculated using unpaired t-test (****p<0.0001 , n.s : non significant). Explants displaying (E) protrusive cells from 10 patients were quantified and results presented as percentage of total explants. Over 50 explants were counted per condition (duplicate) and per patient(Means ± SEM). P values were calculated using unpaired t-test (****p<0.0001 , n.s : non significant). F) Time-lapse sequences of an adenocarcinoma explant undergoing collective invasion into collagen-I gel in the presence of Y27632 monitored by DIC microscopy for 2 days Arrowheads point to non-protruding cells, arrows point to protruding cells and white stars point to lumens. Scale bars: 50 μm.
TFEB ameliorates neuronal loss and neuroinflammation A Immunofluorescence staining of untreated or TFEB‐treated (+TFEB) hippocampus of wild‐type (WT) and rTg4510 (Tau) mice using the anti‐NeuN antibody.Figure 4A Immunofluorescence staining of untreated or TFEB‐treated (+TFEB) hippocampus of wild‐type (WT) and rTg4510 (Tau) mice using the anti‐NeuN antibody. Scale bar: 1000 μm. B Enlarged view of the bracketed areas in A. Scale bar: 50 μm. C Unbiased stereological quantification of NeuN−positive neurons in area CA1 of wild−type (WT) or Tau transgenic mice injected with GFP or TFEB. n = 4 sections/mice, 5 mice/group. Student's t−test, **P = 0.0063. D Wet brain weight measurement of WT or Tau mice with (+TFEB) or without TFEB injection. n = 14 mice/group. ***P < 0.001 (2 way ANOVA with Bonferroni post hoc). Each bar represents average ± s.e.m. E, F Representative immunofluorescence images of cortex (CTX) or hippocampus (HPC) of untreated or TFEB‐treated (+TFEB) rTg4510 (Tau) mice stained with anti‐Iba1 (E) or anti‐GFAP (F) antibodies antibody. Scale bar in E: 200 μm; in F. 400 μm.
The Mpc proteins form dimeric complexes.Figure 2Nickel-affinity-purified Mpc1/Mpc3 hetero-complex used for SEC-MALLS analysis, showing a 1:1 stoichiometry of the protomers. SEC-MALLS analysis of the hetero-complex. The light scattering trace for Mpc1/Mpc3 is shown as a black line. The masses of the protein-detergent-lipid complex (PDL, green), the detergent-lipid micelle (DL, blue) and the protein (P, red) are indicated. Protein fractions across the peak were assessed by SDS-PAGE and visualised by Coomassie Blue staining (B, inset). Nickel-affinity-purified Mpc3 protein used for SEC-MALLS analysis. SEC-MALLS analysis of Mpc3, colour designation as in (B). The protein fractions across the peak (D, inset) were assessed as in (B).
Figure 2A Scheme for the CLIP-UPF1 affinity purification (RIP) assay. B Scatterplot of CLIP-UPF1LL vs CLIP-UPF1SL RIP-seq efficiency. mRNAs were binned according to 3'UTR length (short, medium, or long). C Density plot of recovered mRNAs in CLIP-UPF1LL or CLIP-UPF1SL affinity purifications. mRNAs were binned according to 3'UTR length. Statistical significance was determined by K-S test. D Density plot of recovered mRNAs in CLIP-UPF1LL affinity purifications relative to that of CLIP-UPF1SL. mRNAs were subdivided by PTBP1 and/or hnRNP L motif density within the 3'UTR, as indicated by the gradient triangle. Statistical significance was determined by K-W test, with Dunn's correction for multiple comparisons. E RT-qPCR analysis of indicated transcripts from CLIP-UPF1 overexpression RNA-seq experiments. Relative fold changes are in reference to the GFP expressing control line. Significance of CLIP-UPF1SL or CLIP-UPF1LL overexpression was compared to the GFP expressing control line. Asterisk (*) indicates P < 0.05, as determined by two-way ANOVA. Black dots represent individual data points and error bars indicate mean±SD (n = 3 biological replicates). Dashed lines indicate log2(fold change) of ±0.5. For protected mRNAs, the motif density of PTBP1/hnRNP L within the 3'UTR is indicated. PTC+ indicates the use of primers specific to transcript isoforms with validated poison exons
Figure 2A RFP-ASC specks formation assay of the effect of Ubr2 knockdown on LT-induced inflammasome activation. RAWRA cells were transfected with a control siRNA or Ubr2-targeting siRNA mixtures for 60 h followed by LT treatment. The numbers in the merged panel are the percentages of the cells showing RFP-ASC specks. B Effect of Ubr2 stable knockdown on caspase-1 activation in RAWRA cells. Cells stably expressing a control or Ubr2-targeting shRNA were incubated with WT LT (+) or its E687C mutant (-) for 3 h. C Effects of Ubr2 knockdown (siRNA #05) on LT-induced NLRP1B inflammasome activation reconstituted in 293T cells. Cells were treated with WT LT (+) or its E687C mutant (-). D, E Effect of Ubr2 knockout on NLRP1B or NAIP2-NLRC4 inflammasome activation. WT and Ubr2-/- iBMDMs were treated with LT or BsaK for 3 h. Cell viability was measured by using the ATP assay and data shown are mean values ± SD from three replicates (D).
hnRNPL binds and stabilizes reporter transcripts.Figure 1A The first 400 nt of all 3'UTRs represented in the UPF1 RIP-Seq dataset were analyzed for the occurrence of motifs corresponding to the indicated RNA-binding proteins. Transcripts were divided into groups based on the number of motifs present (0, 1, more than 1), indicated by the gradient triangle. The dotted line indicates the mean RIP-Seq efficiency for all transcripts used in the analysis. Mean and SD are plotted for all groups. B CDF plot of relative abundance of transcripts with 3'UTRs > 1000 nt in K562 cells under conditions of UPF1 or non-targeting shRNA, for transcripts with hnRNP L eCLIP peaks in the indicated intervals Each trace represents a category of CLIP peak locations. For mRNAs with peaks > 400 nt from the TC or with peaks in the 5'UTR or CDS, transcripts with peaks at 3'UTR positions 1-400 were excluded. Statistical significance was determined by Kruskal-Wallis test with Dunn's correction for multiple comparisons and was determined based on comparison to the set with no 3'UTR CLIP peaks. C Representative northern blots assaying the stability of β-globin reporter transcripts containing the SMG5 3'UTR alone or supplemented with cassettes containing the indicated RSE variants in HeLa Tet-off cells. Half-lives were determined from at least 3 independent experiments and decay curves are shown with points representing mean and standard deviation at each timepoint. Significance was measured compared to the SMG5 3'UTR reporter using the sum-of-squares F test. D RNA immunoprecipitation experiments evaluating hnRNP L association with deletion and replacement reporter transcripts from (C). "AS" indicates a reporter in which the antisense sequence of the RSE was inserted in the SMG5 3'UTR in the same position as the RSE. The northern blot is representative of 3 independent experiments and quantification is presented in the accompanying bar graph with mean and standard deviation. * indicates P = 0.0146 in two-tailed Student's t-test. The reporter containing the GAP 3'UTR used here as a transfection control contains a predicted hnRNP L binding site, allowing its consistent recovery in the IPs.