EMBO/sd-character-level-ner huggingface dataset:
Figure 4(A-D) Graphs showing the average number of PH3+ mitotic figures in the posterior lateral pallium of WT and mutant cortices. The total number of mitoses (A) as well as the number of dividing cells in VZ (Pax6+Sox2+ aRGs; B), SVZ (Tbr2+ IPs; C) and outer (basal-most) region of SVZ (oSVZ/IZ; Pax6+ oRGs; D) are shown. For graphs (B-D), statistical analysis by 2-way ANOVA n ≥ 3 brains per age/genotype. Data information: the number of positive cells was quantified in 100µm-width boxes, randomly placed across the lateral pallium. In graphs, data are represented as means ± SEM. 2-way ANOVA *P<0.05, **P<0.01, ***P<0.001). SVZ: subventricular zone; VZ: ventricular zone. (E) Cell cycle duration at different embryonic ages (from E10.5 to E16.5) in WT (blue bars) or KO embryos (orange line), as quantified upon a double EdU/BrdU injection protocol in posterior-most LP n ≥ 3 brains per age/genotype. Data information: the number of positive cells was quantified in 100µm-width boxes, randomly placed across the lateral pallium. In graphs, data are represented as means ± SEM. 2-way ANOVA *P<0.05, **P<0.01, ***P<0.001). (F-G') Ki67 (green; NPs) and EdU (red) IF in WT (F,F') and KO (G,G') after 6 hours consecutive EdU incorporation. Note the higher number of labelled NPs in KO cortices (G') suggesting faster cell cycle progression and/or longer S-phase. Data information: Nuclei (blue) were stained with DAPI. (H) Best linear fit of EdU+Ki67+ NP percentage after 8 hours EdU cumulative labelling in WT (blue line) and KO (orange line) brains. The x- and y-intercept are proportional to the S-phase length and to the number of cycling cells, respectively, while the steepness of the line is proportional to cell cycle duration. WT: y = 11.66x +24.62; KO: y = 13.54x +32.96). n ≥ 2 brains per time point/genotype. Data information: the number of positive cells was quantified in 100µm-width boxes, randomly placed across the lateral pallium. In graphs, data are represented as means ± SEM. 2-way ANOVA *P<0.05, **P<0.01, ***P<0.001). (I-K) PH3 (green; dividing NPs) and Pax6 (red; aRGs) IF of E13.5 WT (I) and KO (J) lateral pallia. Strong and weak/punctate PH3 patterns show M-phase and G2-phase cells, respectively. G2-phase NP percentage is quantified in (K). As a result of shorter G1-phase and globally shorter cell cycle time, the percentage of G2-phase cells is increased in the KO NP pool. n ≥ 2 brains. Data information: Nuclei (blue) were stained with DAPI. the number of positive cells was quantified in 100µm-width boxes, randomly placed across the lateral pallium. In graphs, data are represented as means ± SEM. Student t-test (K; *P<0.05) Scale bars: 50µm. SVZ: subventricular zone; VZ: ventricular zone. (L) Cell cycle time of E13.5 NPs in the lateral pallia of WT (upper column) and KO cells (lower column), as calculated by EdU cumulative labelling and PH3 staining KO NPs have a 16.1% faster cell cycle time, due to a 30.9% shorter G1-phase compensating a 14.3% longer S-phase. n ≥ 3 brains. In graphs, data are represented as means ± SEM. 2-way ANOVA (*P<0.05, **P<0.01, ***P<0.001).
ER expansion in myofibroblasts is coupled to XBP-1 splicing.Figure 3A Analysis of ER size in HFL1 fibroblasts treated with TGFβ in the absence (Ctrl) or presence of the IRE1α inhibitor 4μ8C, or in HFL1 cells transfected with a siRNA control siRNA (siCtrl) or with siRNA targeting XBP-1 (siXBP-1). The ER was visualized with ER tracker Red and nuclei stained with Hoechst (left, scale bars = 20 μm) or by electron microscopy (right, scale bars = 200 nm) where red lines mark the ER.B Quantification of ER size relative to the nucleus from the experimental setup in A. * P = 0.0001 in Ctrl, and * P = 0.0223 in siCtrl. C XBP-1 mRNA expression in untransfected HFL1 cells (UT) and in cells transfected with control siRNA (siCtrl) or siRNA targeting XBP-1 (siXBP-1). * P = 0,0001. D Collagen protein levels in TGFβ treated HFL1 fibroblasts transfected as described in C. * P = 0,005.
Connectome of the zebrafish thyroid gland identifies a dense intercellular signaling network (A) To build a connectome for the atlas, the ligands expressed specifically in each cell-type were matched with their corresponding receptors in the thyrocytes.Figure 5(A) To build a connectome for the atlas, the ligands expressed specifically in each cell-type were matched with their corresponding receptors in the thyrocytes. (B) A highly connected intercellular interaction network is identified by the connectome. The number of ligand-receptor pairs identified between two cell-types is denoted alongside the arrows. For NFE, Gills and Stromal cells, the connectome was restricted to secreted ligands. (C) A dotplot depicting examples of paracrine and autocrine signaling in the thyroid gland. The size of the dots represent percentage of cells expressing the gene in a particular cluster, while the color scale represents average expression of the gene in the cluster after scaling.
a) USP33 depletion inhibits accumulation of LC3–lipid conjugates.figf6(a) USP33 depletion inhibits accumulation of LC3-lipid conjugates. HEK TE cells stably expressing GFP shRNA or USP33 shRNA were incubated with DMEM or HBSS medium for 4 h, in the presence or absence of 50 μM of chloroquine (CQ). Cells were then assayed for the relative accumulation of LC3-I and LC3-II by immunoblotting analysis using anti-LC3 antibody. LC3-II/GAPDH ratios have been calculated using ImageJ densitometric analysis. (b-d) USP33 expression levels affect the accumulation of LC3punctae. Wild-type USP33 or a USP33-shRNA-resistant USP33 mutant (USP33R) was introduced into HEK TE cells stably expressing GFP shRNA or USP33 shRNA. The generated cells were incubated in HBSS medium for 4 h. USP33 was analysed by immunoblotting using anti-USP33 antibody (b). Representative images of LC3immunostaining are shown (c). Endogenous LC3punctae were quantified using the In Cell Analyser 2000 automatic imaging system and IN Cell Investigator software. The mean distribution of LC3punctae per cell is represented as the mean of two independent experiments. Statistics source data for d can be found in Supplementary Table S1 (d). (e) USP33 depletion leads to increased levels of p62. HEK TE cells expressing GFP shRNA or USP33 shRNA were incubated in HBSS medium for the indicated periods of time. Immunoblotting was performed using the indicated antibodies. (f) Overexpression of the RALB-G23V-K47R mutant leads to accumulation of LC3-lipid conjugates. HEK293T cells overexpressing the RALB-G23V or RALB-G23V-K47R mutants were assayed for the relative accumulation of LC3-I and LC3-II by immunoblotting using anti-LC3 antibody.
Figure 2Schematic model describing the domains of MICU1 and the brief structure of the MCU complex green points describing Ca2+ and red points describing mutants. Immunoblots of MICU1 and its mutants rescued by the lentivirus. Arrows indicate MICU1 or the dimer. * indicates the potential DTT-resistant dimer. LDH assay was performed after 16 h of cold stress on ice. Data are presented as mean ± SEM; n = 3, biological replicates. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, n.s.: not significant, by one-way ANOVA followed by Tukey's multiple comparison test Lipid peroxidation was measured by C11-BODIPY 581/591 after cold stress for 5 h. Representative plots are shown in (D). Quantification data (E), whose raw value were normalized by the C11-BODIPY 581/591 of the WT A549 cells after cold stress application, are presented as mean ± SEM; n = 4, biological replicates. ****p < 0.0001, ***p < 0.001, **p < 0.01, n.s.: not significant, by one-way ANOVA followed by Tukey's multiple comparison test.
Figure 5Disruption of cyclin F binding site increased stability of E2F7. E2F7WT and E2F7R894A constructs were integrated in to HeLa/TO system. Cells were arrested with HU for 16 hours before releasing into doxycycline containing medium, and cells were collected every 3 hours for immunoblotting. qPCR showing that mRNA level of E2F7WT and E2F7R894A were at comparable levels. Bar and error bars represent mean ± SEM. Over-expression of E2F7R894A delays cell cycle progression through G2-M phase. HeLa/TO cells expressing either wild-type or mutant version of E2F7 were arrested with 16h of HU, and then cells were released into fresh medium with or without doxycycline. Live cell imaging was performed to trace the G2-M progression of HeLa/TO cells.
The threshold pHi for alkaline‐evoked Ca2+ influx is controlled by VmDepolarization‐evoked Ca2+ signals in sperm mixed with ASW containing high KCl concentrations.Figure 4A Depolarization‐evoked Ca2+ signals in sperm mixed with ASW containing high KCl concentrations. B Ca2+ signals evoked by mixing of sperm with 80 mM KCl and the CatSper inhibitor MDL12330A. C Ca2+ signals evoked by mixing of sperm with 160 mM KCl and MDL12330A. D Dose-response relation for the Ca2+ signals shown in (B, C) at t = 1-2 s. E Threshold pHi for Ca2+ signals evoked by pHi‐clamp solutions in sperm bathed in ASW containing low (3 mM), high (191 mM), and normal (9 mM) KCl (mean ± SD; n ≥ 3); data for 9 mM KCl are from Fig F.F Resting pHi and resting Vm in sperm bathed in ASW containing low (3 mM), high (191 mM), and normal (9 mM) KCl (black) (mean ± SD; n ≥ 3). Mean threshold pHi for CatSper activation at different membrane potentials (red); mean threshold pHi was derived from data shown in (E).
Figure 6We suggest that H3.3-containing nucleosome cores (pink) are more likely to be dynamically exchanged than canonical H3-containing cores (blue) resulting in a greater probability that DNA can become single stranded either as a result of pausing of RNAPII or of transcription-induced negative supercoiling generating transient denaturation bubbles.
The SARS-CoV-2 miR-O7a produces two isoforms processed by DICER and loaded onto AGOs.Figure 3(A) Log2 fold changes of the levels of hsa-miR-let-7a, CoV2-miR-O7a.1 and COV2-miR-O7a.2 in DICER1 knockdown SARS-CoV-2-infected A549-ACE2 cells compared to control cells analyzed by stem-loop RT-qPCR. Results from three independent replicates are shown. (B) In vitro kinetics of DICER1 mediated cleavage of pre-CoV2-miR-O7a stem-loop precursor, pre-CoV2-miR-O7a with mutations that prevent the formation of stem-loop structure and pre-hsa-miR21. Folded conformations for the three precursor molecules are shown. Fold change in the production of mature CoV2-miR-O7a.2 is shown in presence of DICER1 compared to control reaction (no DICER1) as measure by stem-loop RT-qPCR. Bars and error bars represent the average and standard deviation from three independent experiments. (C) Expression levels of the virus-derived miRNAs as a percentage of the viral genome. Absolute quantification of virus-derived miRNAs and viral genome from infected A549-ACE2 cells was performed using two spike-in (see methods). Line represents the average and individual dots represent data from two independent experiments. (D) copy number per cell of hsa-miR-let-7a, CoV2-miR-O7a.1 and CoV2-miR-O7a.2 in infected A549-ACE2 cells quantified using a synthetic small RNA spike-in standard curve by stem-loop RT-qPCR. Levels of hsa-miR-let-7a were normalized for the percentage of infected cells. Bars and error bars represent the average and standard deviation from at least three independent experiments. (E) Loading of hsa-miR-let-7a, CoV2-miR-O7a.1 and COV2-miR-O7a.2 into AGOs as measured by stem-loop RT-qPCR and analyzed as a percentage of input from the immunoprecipitates (IPs) of either pan-AGO IP or control IgG IP from infected A549-ACE2 cells. Levels of miRNA were normalized for the percentage of infected cells.
In vivo function of CNM-causing Dyn2 mutant S619L.Figure 6(A,B) Transferrin receptor (TfnR) uptake, a measure of CME, shown as % uptake of total steady-state surface TfnR for control H1299 cells, and H1299 cells stabily expressing low amounts of siRNA-resistant Dyn2WT-EGFP (A) or Dyn2S619L-EGFP (B) with or without treatment of siRNA-directed towards endogenous Dyn2. (C) Fixed cell TIRF-M images of Dyn2WT-EGFP or Dyn2S619L-EGFP expressing H1299 cells with or without Dyn2-siRNA treatment showing co-localization with clathrin light chain immunostained rabbit anti-CLC and Alexa 547-conjugated secondary antibodies. (D) Percentage of Dyn2-EGFP positive clathrin coated pits detected by live cell TIRF-M and automated master/slave image analysis (Aguet et al, 2013). (E) Lifetime analysis of CCPs which are positive for EGFP-Dyn2WT or EGFP-Dyn2S619L with or without siRNA-mediated specific knockdown of endogenous Dyn2.
MEK and ERK are required for tight junction formation and function.Figure 216HBE cells were seeded sparsely, treated with DMSO, GSK1120212 (500nM), PD0325901 (500nM), or SCH772984 (1M), and incubated for 3 days. All data are representative of 3 independent experiments. (a) Cell lysates were analysed by western blotting for pERK, total ERK, p-p90RSK or total RSK1. 16HBE cells were seeded sparsely, treated with DMSO, GSK1120212 (500nM), PD0325901 (500nM), or SCH772984 (1M), and incubated for 3 days. All data are representative of 3 independent experiments. (b) Cells were fixed and stained for ZO-1 and DNA. Scale bar represents 20m. 16HBE cells were seeded sparsely, treated with DMSO, GSK1120212 (500nM), PD0325901 (500nM), or SCH772984 (1M), and incubated for 3 days. All data are representative of 3 independent experiments. (c) Quantification of tight junction phenotype. >500 cells were counted per sample/experiment, across n=3 independent experiments (dots indicate individual data points). Error bars denote mean ± SEM. ***, p = 0.0002; **** p < 0.0001. (d) 16HBE cells were transfected with control siRNA or siSOS1, and incubated for 4 days. Cell lysates were analysed by western blotting for SOS1, pERK and total ERK. (e) 16HBE cells were seeded on transwell filters with DMSO, GSK1120212 (500nM), or PD0325901 (500nM). Transepithelial resistance (TER) was measured on day 4 and expressed as % of DMSO control. n=3 independent experiments (dots indicate individual data points), error bars denote mean ± SEM. ***, p = 0.0002; **** p < 0.0001. (f) 16HBE cells were seeded on glass-bottomed dishes with DMSO, GSK1120212 (500nM), or PD0325901 (500nM). On day 4, FM4-64 dye was applied to the media and confocal z-stacks acquired. Representative images are shown, the red dotted lines indicate the basal surface. (g) 16HBE cells were fixed and stained for MEK or pERK. Scale bar represents 20m.
miRNAs enriched in Cx43-containing extracellular vesicles are highly structured.Figure 2Modelling of the 3D structure of mature miRNAs enriched in both EVCx43+ (red) and EVCx43- (green). Figure depicts each miRNAs structure, as well as the values of total energy [E(total)], van der Waals energy [E(VDW)] and electrostatic energy [E(elec)].
a) Anti-Ape1 western blot of atg19Δ cells transformed with the indicated expression constructs.figf6(a) Anti-Ape1 western blot of atg19Δ cells transformed with the indicated expression constructs. The lower Ape1 band indicates prApe1 processing and thus its delivery into the vacuole. Rap., rapamycin; Log., (b) Representative electron micrographs of ypt7Δ, atg19Δ yeast cells grown under cytoplasm-to-vacuole targetting (Cvt) conditions expressing the indicated proteins labelled with an anti-Myc antibody. The white arrowheads indicate the isolation membrane. The dashed line indicates the circumference of the prApe1 oligomer. Gold particles, 10 nm. Scale bars, 200 nm. See Supplementary Fig. 5 for full images. (c) Quantification of electron micrographs of yeast cells expressing either Myc-Atg19 or Myc-Atg19-Atg34 co-labelled with anti-Myc (10 nm gold) and anti-prApe1 (5 nm gold). See also Supplementary Fig. 5. Three independent experiments; Myc-Atg19, N = 48, Myc-Atg19-34, N = 24. (d) Anti-Ape1 western blot of atg19Δ cells transformed with the indicated expression constructs. The lower Ape1 band indicates prApe1 processing and thus its delivery into the vacuole. Supplementary Fig. 6 shows the expression of the Myc-tagged proteins and a quantification of the assay. (e) Y2H assay testing for the interaction of Atg11 with Atg19 and the indicated Atg19 mutants. (f) Atg19Δ yeast cells expressing the indicated Atg19 proteins and prApe1-RFP (red fluorescent protein) were labelled with the vacuolar membrane dye MDY-64. Scale bars: 2 μM. The experiments in a-d,f have been conducted three times, the experiment in e twice. Images of uncropped western blots and gels can be found in Supplementary Fig. 7.
Figure 5A. PDGF-AA stimulation resulted in the phosphorylation of WT INVS (top panel, lane 2), but not 3A INVS (top panel, lane 6). PDGF-AA-stimulated INVS phosphorylation is inhibited by Akt inhibitors (top panel, LY294002, MK2206, and GSK690693, lanes 3, 4, and 5, respectively). B. Myr-Akt, a constitutively active form of Akt, was expressed together with WT or 3A INVS in HEK293 cells and INVS phosphorylation was quantified. WT, but not 3A INVS, can be phosphorylated in the presence of Myr-Akt. C. 3A INVS exhibited a weaker interaction with Akt as compared to WT INVS (top panel, compare lanes 1 and 2, WT and phospho-defective mutant, respectively). Expression of Akt and INVS are shown underneath.D. 3A INVS exhibited weaker dimerization as compared to WT INVS (top panel, compare lanes 1 and 2, WT and 3A, respectively). Expression of Akt and INVS are shown below each. E. Length of primary cilia in confluent hTERT-RPE cells was analyzed after siRNA-mediated knock down of Akt1/2. Ciliary length decreased in cells expressing siRNA. Results presented are means ±SE of the longitudinal length of acetylated-tubulin (red), a marker of primary cilia, in siRNA-transfected (green) hTERT-RPE1 cells (n=175 for control siRNA and Akt siRNA, and n=195 for Akt siRNA plus siRNA resistant Akt, respectively). Three independent experiments showed similar results. Statistical significance was analyzed by student's t test. Note that re-introduction of siRNA-resistant Akt (Matsuda-Lennikov et al., 2014) in Akt knockdown cells rescued the effect on ciliary length. F. Expression of 3A INVS (bottom panels) but not WT INVS (middle panels) inhibited the development of primary cilia as compared to control cells (top panels). Quantification of ciliary length. Results are mean ± SE of longitudinal length of acetylated-tubulin (blue), a marker of primary cilia, in EGFP-INVS (green) transfected in hTERT-RPE1 cells (n=58 for EGFP vector and for EGFP-INVS WT, and n=52 for EGFP-INVS 3A mutant, respectively). Three independent experiments showed similar results. Statistical significance was determined by student's t test.
VP1388 and VP1390 are loaded together on the T6SS spike and are secreted co-dependently.Figure 3A VP1388 binds VP1390. Immunoprecipitation using α-FLAG antibodies from E. coli cells co-expressing the indicated C-terminal FLAG- and Myc-tagged proteins from arabinose-inducible plasmids. B VP1388 and VgrG1 co-precipitate with VP1390. Immunoprecipitation using α-FLAG antibodies from V. parahaemolyticus ∆hns/∆hcp1/∆vp1390 derivatives harboring plasmids for the arabinose-inducible expression of FLAG-tagged sfGFP or VP1390. Cells were grown in MLB media supplemented with chloramphenicol to maintain the plasmids, and 0.1% arabinose. Endogenous VP1388 and VgrG1 were detected using α-VP1388 and α-VgrG1 antibodies, respectively. C Expression (cells) and secretion (media) of VP1388, VP1390, and VgrG1 from the indicated V. parahaemolyticus ∆hns-derived strains harboring an empty plasmid (pEmpty) or plasmids for the arabinose-inducible expression of VP1388 (pVP1388) or VP1390 (pVP1390). Samples were grown in media containing 3% NaCl and supplemented with 0.1% arabinose at 30°C. RNA polymerase β (RNAp) was used as a non-secreted protein loading control. Arrows denote the bands at the expected size of VP1388 or VP1390, as indicated in the appropriate panels.
Co-expression of factors activated late in EpiSC resetting increases pluripotency marker expression and significantly reduces the resetting time scale.Figure 4(A) Top: Predictions of the number of regulation steps required for full activation of the indicated gene under control or dual expression of Esrrb and Tbx3 (+E/T). Bottom: Gene expression of EpiSCs harbouring empty vector (grey) or Esrrb/Tbx3 (orange), capture at D0 (F/A), D2 and D4 (as described in Fig 3D). Dashed black line: expression levels in ESCs maintained in 2i+LIF. Data normalised to empty vector cultures in F/A. Gapdh serves as an internal control. Mean +/- SEM, n=3 independent experiments. (B) As for panel A, comparison of control with dual expression of Esrrb and Klf4 (+E/K, green in bottom plot). Mean +/- SEM, n=3 independent experiments for bottom qRT-PCR panels (C) Left: Flow cytometry profiles of the resetting progression of EpiSCs stably transfected with the Esrrb-T2A-Klf4 construct and cultured in 2i+LIF with DOX for 2 and 4 days, with the indicated fraction of cells sorted for colony formation assay. Since the Venus reporter is under the control of a DOX responsive element, and the emission spectra of Venus and GFP fluorescence overlap, Oct4-GFP reporter could not be fully distinguished from Venus expression. Right: Number of AP+ colonies formed from 250 sorted cells from indicated fractions. Data points represent two technical replicates of one out of two independent experiments (D) Heatmap of single-cell expression measured by qRT-PCR of major ESC and EpiSC markers in un-induced EpiSCs (black), established ESCs (red), Day 2 High/Low (dark and light blue), and Day 4 High cells (green) (E) Scatterplots of the relative expression of pluripotency and transitional markers. Red bar corresponds to median gene expression, and each dot represents a single cell
a–c, RFA1::FLAG DDC2::MYC cells were arrested in G2 and released in YP galactose to induce HO endonuclease.f1a-c, RFA1::FLAG DDC2::MYC cells were arrested in G2 and released in YP galactose to induce HO endonuclease. After 30 min, the culture was split in two: +VPA and -VPA. a, Samples were processed for western blot using anti-Rad53, Srs2 and Ddc2 antibodies. a-c, RFA1::FLAG DDC2::MYC cells were arrested in G2 and released in YP galactose to induce HO endonuclease. After 30 min, the culture was split in two: +VPA and −VPA. b, A schematic diagram showing probe locations with respect to the HO cut site. The resection rate was calculated as the rate of HO cut band disappearance. a-c, RFA1::FLAG DDC2::MYC cells were arrested in G2 and released in YP galactose to induce HO endonuclease. After 30 min, the culture was split in two: +VPA and -VPA. c, Fold enrichment of the 0.2-kb fragment was calculated after ChIP of Rfa1-Flag, Ddc2-Myc. d, VPA effect on ectopic recombination (rec.) in wild-type (WT) cells. Error bars represent standard deviation (s.d.) calculated from four independent experiments.
Seroconversion of patients during baricitinib treatment.Figure 4Detection of IgG antibodies against the S1/S2 antigens of SARS-CoV-2. Time of baricitinib treatment is indicated for all patients (patients A, B, and D for 10 days, patient C for 12 days).
Figure 2A-B. Analysis of DCC (A) and netrin-1 (B) expression in a cohort of normal tonsils (n=9), Germinal-Center-B-Cell-like-DLBCL (n=13), Activated-B-Cell-like-DLBCL (n=18) and MCL (n=36). Quantification of genes expression was performed by Q-RT-PCR, relatively to HPRT housekeeping gene. **: p<0.005; ****: p<0.0001; two-sided Mann-Whitney U-test. C-D. Analysis of netrin-1 expression in DLBCL (C) and MCL (D) by immunohistochemistry. C. 21 DLBCL patients biopsies were used to build a TMA. Normal tissues (normal tonsils, reactive lymph nodes, muscle, liver) are included (the left-most spot, upper panel, corresponds to liver). Enlarged representative image of netrin-1 expression in DLBCL is shown (lower panel). D. Representative images of netrin-1 expression in paraffin-embedded biopsies of MCL (n=9). Five tumors were positive for netrin-1 (left panel) and four were negative (right panel).