EMBO/sd-character-level-ner huggingface dataset:
a,b) Association of EEA-1 (a) or Rab5 (b) with phagosomes generated in primary macrophages by 3-μm beads coated with E.figf4(a,b) Association of EEA-1 (a) or Rab5 (b) with phagosomes generated in primary macrophages by 3-μm beads coated with E. coli outer membrane extract; numbers in bottom corners indicate time (in min). α-, anti-. Original magnification, ×60. Right (a,b), quantification of fluorescence. Data are representative of at least three independent experiments (error bars, s.d.).
Cyclin D1b expression sensitizes cells to senescence induced by therapeutic challenge.Figure 7Ccnd1+/+ and Ccnd1KI/KIMAF lines were grown in serum-proficient media for 24 h. Cells were then treated with control DMSO or 2.5 μM of the PARP inhibitor ABT-888. One hour post-treatment, the indicated lines were treated with 5 Gy of radiation. Cells were then allowed to recover for 48 h, after which BrdU was added for 1 h, and then harvested for bivariate flow cytometry. Representative traces for each condition are shown (left) and BrdU incorporation of biological triplicates was quantified (right). Cells were plated and treated as in (A). Forty-eight hours post-treatment, cells were fixed and stained for markers of senescence (β-galactosidase activity). Cells positive for the staining (blue) were quantified for each condition (400× magnification) and reported as a percentage of the total population (right). Plates treated in parallel with both IR and ABT-888 were harvested at 48 h via 1× trypsin and re-plated in serum-proficient media. Cells were allowed to grow for 96 h and were then stained with crystal violet (4× objective, with inset at 200× magnification, boxes highlight area of magnified images).
A mathematical model that includes area dependence and gene-specific correlation in burst size captures the observed time-dependent bivariate mRNA distributions.Figure 3(A) Schematic of the five different model features (F1-F5) used in the four models considered (see Table 1). F1: assumes the burst frequency oscillates over 24 hours; F2: assumes a fixed burst size for each gene. The burst frequency (f) and burst size (b) determine the shape of the NB distribution used to model the mRNA counts (see Methods), where the mean μ = bf and the variance σ2= μ + μ2/f. F3: the burst size in each cell scales with measured cell area using a gene-specific exponent βg. F4: to model incomplete synchronisation, each cell has a cell-specific phase offset compared to the average phase. F5: the burst size in each cell is proportional to the cell area multiplied by a cell-specific burst parameter εi, g. These cell-specific εi, g are further modelled using a bivariate log-normal distribution. (B) PSIS-LOO calculated for each of the four models using the whole dataset of both gene-pairs across all time points. (C) Posterior parameter estimates. Burst frequency is plotted as a function of time, where the solid line represents the posterior mean and the shaded area represents the 90% confidence interval. Posterior probability densities are shown for the average burst size, β (which controls the dependence between burst size and cell area) and ρ (representing the correlation in burst size between genes (in log space)), where β is inferred from model M2 and the average burst size and ρ are calculated from model M4. (D) Comparison of the probability density of the data (kernel density estimates) with the model (using model M4). To estimate the probability from the model, the data set was simulated 15 times using the posterior mean parameter values combined with the measured cell areas.
a–d) Fluorescence images of GFP-p38, -ANK1-p38 and -NA Sph1 Agm in SY5Y cells immunostained for endogenous beclin-1 (a), Vps34 (b), Atg14L (c) and DFCP-1 (d).f6(a-d) Fluorescence images of GFP-p38, -ANK1-p38 and -NA Sph1 Agm in SY5Y cells immunostained for endogenous beclin-1 (a), Vps34 (b), Atg14L (c) and DFCP-1 (d). Arrows indicate colocalization.
Figure 8A-D. Primary mouse bone marrow-derived macrophage cells (BMDM) from adult male WT-mLRRK2 BAC transgenic mice were immunostained with anti-mouse CCR5, or CD11b, or MHC II antibody (shown as green signal) and (A) total Rab10 antibody (red signal), or (B) pT73-Rab10 antibody (red signal) as indicated. Representative photomicrographs (from >20 images analyzed for each condition from n=3 biologically independent experiments) are shown. (C) Photomicrographs showing the different cargo with respect to Rab10 tubular structures. (D) EEA1 (shown as green signal) with total Rab10 or pT73-Rab10 (shown as red signals) as indicated. Bounding boxes in panels A-D are magnified in "High Mag" panels that show individual vesicles. Scale bars represent 10 μm and 2 μm for "High Mag" images. E. Calculated percentage of vesicles positive with tubular structures (n>30 vesicles analyzed from cells from n=3 biologically independent experiments) are shown. Each dot represents one vesicle, with significance assessed by Mann-Whitney test. ** represents P<0.01. Error bars represent ± SEM. F. BMDM cells were treated with 0.004% DMSO or 100 nM MLi2 prior to live cell surface staining with Alexa-594 conjugated rat anti-mouse CD11b antibody on ice for ten minutes. Three wells of cells from each condition were fixed and imaged for initial surface CD11b binding (shown in red as indicated). Six wells of cells from each condition were further incubated at 37°C to allow rapid internalization of CD11b-antibody complexes for ten minutes prior to striping of surface CD11b antibody using 0.5% acetic acid on ice for one min. Three of the six wells were fixed and surface-stained with Alexa-488 conjugated goat anti-rat antibody to show CD11b-antibody complexes remaining on the cell surface (shown in green as indicated). Internalized CD11b-antibody complexes are shown in red. The other three wells of cells were further incubated at 37°C again for ten min to allow recycling of internalized CD11b-antibody complex followed by surface staining with Alexa-488 conjugated goat anti-rat antibody to show CD11b-antibody complexes recycled back to the surface. Representative images from n=3 biologically independent experiments are shown. Scale bars represent 30 μm. G-I. (G) The initial surface CD11b, or (H) internalized CD11b, or (I) recycled CD11b were calculated from >20 images analyzed for each condition from n=3 biologically independent experiments. Each dot represents the mean value from one experiment. Error bars show ± SEM with significance assessed by Mann-Whitney test. * represents P<0.05.
Combining macrophage depletion with chemotherapy increases survival of glioma bearing mice.Figure 8A. Experimental design for the survival trial: 8 weeks old wild type mice were injected with GL261 cells to induce glioma formation and were assigned randomly to vehicle (n = 6), anti-CSF1 monoclonal antibody treatment (0.5mg ; n=6), temozolomide treatment (40mg/kg ; n=6) or temozolomide and anti-CSF1 mAb combination treatment (n = 6). Mice were weighed once daily until they developed symptoms or reached the trial endpoint. B. Survival curves of the mice described in A. Combination therapy is significantly more efficient than chemotherapy alone. (n=6 mice per group). Statistical analysis: B. Multiple comparisons Mantel-Cox log-rank; Error bars: meanSD. Scale bars: 300µm C. phospho-H2AX and Glut1 immunohistochemistry on 3 weeks growth glioma in wild-type mice treated as described in A. Anti-CSF1 antibody in combination with temozolomide chemotherapeutic agent induces a wider tumor cell death efficiency with a significant decrease of blood vessel caliber (D.) (50µm depth stack) (n=6 mice per group). Statistical analysis: D.E. one-way ANOVA followed by multiple comparisons Tukey's test. Error bars: meanSD. Scale bars: 300µm Anti-CSF1 antibody in combination with temozolomide chemotherapeutic agent induces a wider tumor cell death efficiency with an increase in caspase3 positive tumor cells (E.) (50µm depth stack) (n=6 mice per group). Statistical analysis: D.E. one-way ANOVA followed by multiple comparisons Tukey's test. Error bars: meanSD. Scale bars: 300µm
Figure 8(A) Gating strategy for the sorting of freshly purified resting human CD4+ T cells based on CENP-A expression. Dead cells were excluded (first plot) using Fixable Viability Dye eFluor 780, CENP-Ahigh and CENP-Alow cells (both CENP-B+) were sorted (second plot). Post-sort analysis of each population is then shown (third and fourth plots). (B) Representative images of sorted CD4+ T cells with high or low CENP-A level. The nucleus is contoured by a white dashed line. Scale bar, 5 µm. (C) Schematic representation of the analysis of CENP-A expression over time in CD4+ T cells, shown in D-G. T cell were activated with anti-CD3/anti-CD28-coated beads. (D) Tracking of cell division by CFSE dilution. The black line separates cells that have not divided (nº of div=0, CFSE-high) from those that have undergone at least one division (nº of div≥1, CFSE-low). (E) CENP-A expression and CFSE dilution. Black lines were set based on CENP-A isotype control and CFSE maximal staining at day 0 (cells in shaded quadrants are CFSE-high/CENP-A-low). One representative donor is depicted. (F) Absolute number of CFSE-high/CENP-A-low CD4+ T cells (shaded gate in E). Symbols represent individual donors. Error bars represent SEM from 6 independent donors. One-way ANOVA, multiple comparisons, n=6. *p < 0.05; **p < 0.01. (G) Frequency of cells without centromeric CENP-A determined by immunofluorescence microscopy before and post activation. Symbols represent individual donors, n=4. Error bars represent SEM from 6 independent donors. (H) Model of the role of CENP-B in the maintenance of centromere identity. I-III: See text for details. In III: (top) representative image of a pseudo-dicentric chromosome in PD-NC4 cells with an active (red) and inactive (green) centromere bound by CENP-C and CENP-B, respectively; (bottom) example of a rare centromere re-activation in which the inactive (red) centromere gains CENP-C binding capacity. White dashed lines contour representative chromosomes. Schematic to the right illustrate the observed CENP-C and CENP-B localization patterns on a chromosome.
Composition of oskar mRNPs in situ.Figure 5(A, B, C) Confocal image of a Tm1eg9 homozygous egg-chamber expressing EmGFP-Tm1-I (green) and Khc-mKate2 (magenta). oskar mRNA labelled with osk1-5 FIT probes(Hovelmann et al., 2014) is in yellow.(B', C') oskar mRNPs co-localizing with both EmGFP-Tm1-I and Khc-mKate2. Colors indicate the maximal co-localization distance (C'). Panels B-C' represent the boxed regions in panel A. (D, E) Localization of Khc-mKate2 (green) and oskar mRNA (magenta) in wild-type and Tm1gs mutant nurse cells.(F, G) Fraction of oskar mRNPs co-localizing with Khc-mKate (dark grey), EmGFP-Tm1-I (white), or both of these proteins (light grey) in the oocyte (F) or in the nurse cells (G) (max. colocalization distance is 250 nm). None of the values are significantly different from each other (one-way ANOVA, p>10-3). Horizontal dashed lines indicate the expected value of observing both proteins in an oskar mRNP if the interactions are independent (see Fig EV3C and D). Significance of the observed co-localization values versus the expected values are shown.(H) Fraction of oskar mRNPs co-localizing with Khc-mKate in wild-type and Tm1gs mutant nurse cells when half or all Khc molecules are labelled (as indicated above the graph). P values of pair-wise t-tests are indicated.(I) Fraction of oskar mRNPs co-localizing with free mCherry in wild-type nurse cells used as negative control. The measured fraction (~1.6%) is significantly different from zero (one sample t-test). All other measured co-localization values are significantly different form this negative control (p<0.001, one-way ANOVA).(F-I) Numbers indicate the number of particle clusters (100 oskar mRNPs in each) and the number of egg-chambers (in brackets) analysed. Error bars represent 95% confidence intervals. All values (F-I) are significantly different from zero (p<10-3, one sample t-test).
Figure 1Figure 1. Comparison of genome structures recovered from five synchronized stages over the cell cycle.(A) Overview of the different synchronization time-points with corresponding FACS profiles and representative images of DAPI-stained cells. Figure 1. Comparison of genome structures recovered from five synchronized stages over the cell cycle. (B) 3D average representation of the Hi-C contact map of a yeast G1 population. The color code reflects chromosomal arm lengths and, centromeres, telomeres and rDNA are highlighted. Figure 1. Comparison of genome structures recovered from five synchronized stages over the cell cycle. (C, D) Comparison of contact maps. The 16 yeast chromosomes are displayed atop the maps. Black arrowheads: inter-telomere contacts. Yellow arrowheads: inter-centromeric contacts. Left panels: Hi-C maps obtained from two G1 cell populations synchronized independently (C) . Right panels: log-ratio between each pair of maps. Insets display magnifications of chr4. Blue to red color scales reflect the enrichment in contacts in one population with respect to the other (log2). Figure 1. Comparison of genome structures recovered from five synchronized stages over the cell cycle. (C, D) Comparison of contact maps. The 16 yeast chromosomes are displayed atop the maps. Black arrowheads: inter-telomere contacts. Yellow arrowheads: inter-centromeric contacts. Left panels: Hi-C maps obtained from G1 and G0 populations (D). Brown to yellow color scales reflect high to low contact frequencies, respectively (log10). Right panels: log-ratio between each pair of maps. Insets display magnifications of chr4. Blue to red color scales reflect the enrichment in contacts in one population with respect to the other (log2). Figure 1. Comparison of genome structures recovered from five synchronized stages over the cell cycle.(E) Pairwise Euclidian distances between contact maps of populations of G0, G1 synchronized either with elutriation or blocked using a cdc6 mutant, metaphase (cdc20 mutant), and anaphase (cdc15 mutant) cells. Color code: contact map similarity. Figure 1. Comparison of genome structures recovered from five synchronized stages over the cell cycle. (F) Principal Component Analysis (PCA) of the distance matrix in (E).
DDR activation induces ACE2 expression in cultured cells and in vivo.Figure 3A Immunofluorescence showing gammaH2AX foci in MEFs Trf2F/F at the indicated time points following 4OHT treatment and consequent TRF2 knockout. Scale bar, 25 µm. B RT-qPCR detection of Ace2 mRNA expression levels in MEFs Trf2F/F treated as in A (n = 3 independent experiments). C Immunofluorescence showing gammaH2AX foci in HeLa shTRF2 cells at the indicated time points following doxycycline treatment and consequent TRF2 knockdown. Scale bar, 25 µm. D RT-qPCR detection of ACE2 mRNA expression levels in HeLa shTRF2 cells treated as in C (n = 6 independent experiments). E Representative immunofluorescence images of 53BP1 staining in liver from Trf2F/F mice treated with tamoxifen (to induce TRF2 loss and telomere uncapping) or vehicle. The animals as been injected also with PBS. Scale bar, 10 µm. F RT-qPCR detection of Ace2 mRNA expression levels in livers of mice treated as in E (n = 5-7 mice per group).
miR-105 and miR-204 in cancer-secreted EVs target RRAGC.Figure 5A. Western blot analysis of CAF and NIH3T3 cells transfected with indicated anti-miRNAs or control for 24 h and then treated with EVs or PBS for 48 h. B. Sequences of the predicted binding sites for miR-105 and miR-204 in the 3'UTR of human RRAGC. C. Dual luciferase assay of the psiCHECK reporters containing the 3'UTR of RRAGC that was wild-type, with all three miR-105 sites mutated, or with the miR-204 site mutated. NIH3T3 cells were co-transfected with the reporter and indicated miRNA mimic (n=3 biological replicates for each group). D. Dual luciferase assay of the indicated psiCHECK reporters in NIH3T3 cells treated with EVs or PBS for 48 h (n=3 biological replicates for each group).
Figure 2A, B Representative micrographs of H&E stained histological sections of xenografted mammary gland from 3 female mice 1 month after injection of MM134 (A) or SUM44 cells (B). Scale bars, 100 μm. C Representative micrographs of H&E stained histological sections of MM134 xenografts from 3 different female mice 5 months after intraductal injection. Arrows point to indian files. Scale bars, 100 μm. D Representative micrographs of H&E stained histological sections of SUM44 cell xenografts from 3 different female mice 5 months after intraductal injection. Arrows point to indian files. Scale bar, 50 μm. E, F Representative micrographs of IHC for Ki67, ER, and PR on histological sections of MM134 (E) and SUM44 (F) xenografts 5 months after intraductal injection counter stained with hematoxylin. Scale bars, 200 μm. Bar plots represent means ± SEM of measurements and indicate the percentage of positive cells for >1000 cells counted on 3 glands. G Representative micrographs of PR-IHC on glands from 5-month-old MM134 xenografts from hosts treated for 2 months with vehicle (left) or E2 (right) pellets. Scale bars, 200 μm. Right, bar plot showing percentage of PR+ cells in MM134 and SUM44 xenografts with n>1000 cells per condition on glands from 3 different mice. Data represent mean ± SEM unpaired, Student's t-test, two-tailed; *** p < 0.001, **** p < 0.0001. H Fluorescence stereomicrograph of a mammary gland of a NSG:EGFP+ mouse 2 months after injection with MM134-RFP/luc2 cells. Scale bar, 1 mm. I, J Micrographs of IF for GFP and E-cadherin (I) and GFP and SMA (J) counterstained with DAPI of MM134 glands 1 month after intraductal injection of NSG:EGFP+ mice, n=4. Scale bars, 50 μm. K Scheme showing the timeline of lobular carcinogenesis in the intraductal model.
Figure.Figure 4A. MEFs were treated with 10 μg/ml FAC for the indicated times and harvested. Cell lysates were fractionated and analyzed by SDS-PAGE with the indicated antibodies. Asterisks indicate a non-specific band. TfR1; Transferrin receptor 1 B. MEFs were cultured for 12 h in medium containing 10 μg/ml FAC supplemented with DMSO or 10 μM E64d and 10 μg/ml pepstatin A (pepA). Samples were analyzed by immunoblotting with the indicated antibodies. The black arrowhead indicates an intact ferritin band, and the white arrowhead indicates a partial degradation band of ferritin. C. NCOA4 KO MEFs reconstituted with hNCOA4-myc were cultured with 10 μg/ml FAC for the indicated times and fixed for imaging. Cells were immunostained with ferritin and myc antibodies. Scale bar, 10μm. D. Myc (NCOA4) puncta were extracted, and average signal intensities of myc and ferritin in the puncta in (C) were plotted. E. MEFs expressing hNCOA4-TurboID were treated with 10 μg/ml FAC for 6 h or 24 h and then cultured with DMSO or 50 μM biotin for 30 min. Cells were lysed with Triton buffer, and lysates were pulled down by streptavidin beads. Inputs and pulldown samples were analyzed with immunoblotting by indicated antibodies. F. WT MEFs pretreated with 10 μg/ml FAC for 6 h or 24 h were chased with 20 μg/ml CHX and analyzed by immunoblotting with the indicated antibodies. G. NCOA4 KO MEFs reconstituted with myc-hNCOA4 were pretreated with 10 μg/ml FAC and then cultured with 20 μg/ml CHX or 200 nM bafilomycin A1 (BafA). Cells were fixed for imaging and immunostained using myc and LAMP1 antibodies. Scale bar, 10μm.
Sema3C inhibits vessel formation in a xenotransplantation model.Figure 2Scheme of the experimental procedure. Pericytes were lentivirally transduced with Sema3C or GFP expression vectors. HUVEC/pericyte spheroids (1:1 ratio) were injected s.c. into SCID mice. Microvessels within the plugs stained against human-specific CD34. Cell nuclei were stained with DAPI. Scale bar, 100 μm.The area of CD34-positive microvessels was quantified and normalized to the total area of plug. Results represent the means ± s.e.m of 5 plugs (control) and 7 plugs (Sema3C), *P = 0.018. Vessel coverage was assessed by αSMA staining. Scale bar, 100 μm.Vessel coverage was quantified as the ratio of endothelial cell-associated mural cells to total amount of vessels (n = 5 (control) and n = 7 (Sema3C)).Vessel perfusion was evaluated by i.v. injection of FITC-lectin (0.15 mg) 30 min before mice were sacrificed. The data show the percentage of perfused vessels in the plug and normalized to total vessel area (n = 5 (control) and n = 7 (Sema3C)).
Figure 7A. Representative confocal images from EGFP-GSK3α FRAP experiments in organotypic slices cultured from C57 wild-type mice. Upper left panel ('Baseline') shows EGFP-GSK3α expression in a dendrite and dendritic spine. The spine was bleached ('Bleach'; yellow dashed circle) and its fluorescence partially recovered 90 s later ('Recovery'). Single dendritic spines were imaged once per second for 10 seconds prior to, and for 90 seconds following, spine photobleaching. Such single FRAP experiments were performed on different spines during basal conditions ('basal'; upper panels), immediately after cLTD induction with 20 µM NMDA for 5 minutes ('cLTD'; middle panels), and immediately following 10 minutes of washout ('washout'; lower panels). Scale bars = 1 µm. B. Time course of fluorescence recovery during FRAP experiments described in A. White circles ('basal') show the average spine/dendrite fluorescence of FRAP on EGFP-GSK3α-expressing spines in wild-type mouse slices prior to cLTD induction. Pink circles ('cLTD') show fluorescence in spines in which cLTD had been induced using 20 µM NMDA. Green circles ('washout ') represent the average of spine fluorescence after NMDA washout for 10 minutes. Recovery trajectories for the three conditions are fitted with two-phase exponential curves (red lines). C, D. As A, B but in slices cultured from C57 tauKO mice. Scale bars = 1 µm. E. Quantification of last 10 seconds of fluorescence recovery (90-100 seconds) in B and D. Error bars represent SEM and statistical significance was calculated according to the Mann-Whitney U test. N = 13, 11 and 12 spines for wt baseline, cLTD and recovery and 9, 7 and 5 spines for tauKO baseline, cLTD and recovery groups, respectively. F. Spine/dendrite ratios showing enrichment of EGFP-GSK3α in wild-type or tauKO slices in 'baseline', 'cLTD' or 'recovery' conditions. Error bars represent SEM and statistical significance was calculated according to the Mann-Whitney U test.