EMBO/sd-character-level-ner huggingface dataset:
Fig.Figure 1Gata6-WT and Gata6-KOmye peritoneal macrophages (pMɸ) were unstimulated (-) or stimulated with TLR2L Pam3CSK4 (500 ng.ml-1), TLR3L Poly I:C (1 µg.ml-1), TLR4L purified LPS (100 ng.ml-1), TLR5L Flagellin (100 ng.ml-1), TLR7 and 8L R848 (1 µg.ml-1) or TLR9L CpG ODN1826 (5 µM). Culture supernatants were collected 24 h after the start of stimulation and IL-1β and TNF ELISA were performed. n=5, two-way ANOVA analysis with Tukeys multiple comparison post-test. IL-1β (C) and TNF (D) ELISA of Gata6-WT and Gata6-KOmye pMɸ stimulated for 24 h with the indicated LPS concentrations. n=3, two-way ANOVA analysis with Sidak's multiple comparison post-test. IL-1β (E) and TNF (F) ELISA from Gata6-WT and Gata6-KOmye pMɸ stimulated with LPS (100 ng.ml-1) for the indicated times. n=3, two-way ANOVA analysis with Sidak's multiple comparison post-test. IL-1β ELISA of Gata6-WT and Gata6-KOmye pMɸ stimulated for 18 h with 100 ng.ml-1 LPS or recombinant IL-1 receptor antagonist (rIL-1ra), n=4 to 5, two-way ANOVA analysis with Tukey's multiple comparison post-test. IL-1β ELISA of Gata6-WT and Gata6-KOmye pMɸ stimulated for 18 h with 100 ng.ml-1 LPS or 100 ng.ml-1 recombinant TNF (recTNF), n=4 to 5, two-way ANOVA analysis with Tukey's multiple comparison post-test.
Figure 2Mass-spectrometry analysis of proteins purified from extracellular matrix isolated from H1299control and H1299RASSF1A lung adenocarcinoma cell lines with summary of results. Only proteins identified with two or more peptides were taken into consideration. The resulting list of proteins was restricted to ECM proteins (n=3). Kaplan Meier plots depicting prognosis in TCGA_LUAD (lung adenocarcinoma) with high and low mRNA expression of ECM proteins revealed by proteomics. Significance derived from log-rank test. Data information: Unless otherwise indicated all statistical analyses were performed using Student's t-test (2-tailed) of n=3 experiments and error bars represent the mean ± S.E.M. Representative images of H&E and IHC staining for P4HA2, and quantification (bars) of two independent regions of n=5 H1299control and n=3 H1299RASSF1A primary lung tumours. Scale bars 100µm. Data information: Unless otherwise indicated all statistical analyses were performed using Student's t-test (2-tailed) of n=3 experiments and error bars represent the mean ± S.E.M. Hydroxyproline assay (Cell biolabs Inc.) for total hydroxyproline activity of cells. Data information: Unless otherwise indicated all statistical analyses were performed using Student's t-test (2-tailed) of n=3 experiments and error bars represent the mean ± S.E.M. Western blot indicating levels of Collagen I in H1299control and H1299RASSF1A cells. Western blot indicating levels of Collagen I in H1299control cells in the presence of (4μM) prolylhydroxylase inhibitor (DPCA), siRNA against P4HA2 or combination. Quantification of immunofluorescence staining of Collagen I in H1299control cells in the presence of (4μM) prolylhydroxylase inhibitor (DPCA), siRNA against P4HA2 or combination. H1299RASSF1A cell line was used as negative control for comparison. Data information: Unless otherwise indicated all statistical analyses were performed using Student's t-test (2-tailed) of n=3 experiments and error bars represent the mean ± S.E.M. Immunofluorescence staining for Collagen I and P4HA2 and quantification (right graph) in HOP92 cells after siRNA against RASSF1A. Scale bars 20µm. Data information: Unless otherwise indicated all statistical analyses were performed using Student's t-test (2-tailed) of n=3 experiments and error bars represent the mean ± S.E.M. Western blot analyses in HOP92 cells showing increased expression of collagen I after siRNA against RASSF1A. Immunofluorescence staining for Collagen I and P4HA2 and quantification (right graph) in HeLa cells after siRNA against RASSF1A. Scale bars 20µm. Data information: Unless otherwise indicated all statistical analyses were performed using Student's t-test (2-tailed) of n=3 experiments and error bars represent the mean ± S.E.M.
Structure of Rab-specific PPM1H phosphatase.Figure 1(A) Domain organization of PPM1H, PPM1J, and PPM1A. The annotation of regions (anchor, flap domain) is discussed in the text. The loop deletion (188-226) that was engineered to improve diffraction is indicated. (B) Ribbon model of the enzyme with a view to the catalytic cleft that contains two Mg2+ ions (cyan spheres). The N-terminal region is magenta (33-71), the flap domain is a wheat colour, and the β-sheet motif is green. The loop deletion (188-226) connects α1/α2 on the opposite face relative to the active site. The back view of the enzyme is also shown with a 180° rotation around the axis indicated. Parts of the anchor (RPxFL motif, magenta) that interact with the globular core are shown as stick models, and discussed in the text. The β1 strand is orange to emphasize its non-canonical conformation due to the presence of the preceding anchor. (C) Comparisons of the flap domain of PPM1H with PPM1A (left) and the tudor domain (right). Apart from a conserved loop (dotted circle) which forms an interface with the catalytic domain, the sequences and structures of flaps are diverse among the PPM family. (D) The indicated amounts of recombinant wild-type and mutant PPM1H or PPM1J (with a His-Sumo N-terminal tag, expressed in E. coli) were incubated in vitro with 2.5 µg pThr72 phosphorylated Rab8a (left) or pThr73 phosphorylated Rab10 (right) for 20 min in the presence of 10 mM MgCl2 in 40 mM HEPES pH 7.5 buffer. Reactions were terminated by addition of SDS Sample Buffer and analyzed by Phos-tag gel electrophoresis that separates phosphorylated (slow migrating) and dephosphorylated Rabs. The gel was stained with Instant Blue Coomassie. D288A is a substrate-trapping (inactive) variant of PPM1H and was used as a control.
Figure 1Schematic diagram depicting the procedure of compound screening during mouse chemical reprogramming. The chemical screening result evaluated by GFP+ colony number on d24. GFP+ colony number of samples treated with R406, R788 and Lenvatinib. n = 3. Fluorescence image of colonies with pOct4-GFP expression after DMSO and R406 treatments. Scale bar, 100 μm. Concentration (F) and stage (G) test of R406 (E) during reprogramming. n = 3. Immunofluorescence of early pluripotent marker Sall4 in DMSO- and R406- treated cells on d12. n = 5. Scale bar, 100 μm. RT-qPCR analysis of Sall4, Cdh1, Epcam and Esrrb gene expression in MEFs, intermediate cells on d12 treated with DMSO and R406, and R1 (mESCs). n = 3. Diagram showing the procedure of shSyk virus infection at the early stage of reprogramming. RT-qPCR analysis of Syk expression in MEFs infected with shNC and shSyk viruses. n = 3. Immunofluorescence of Sall4 in reprogramming intermediates infected with shNC and shSyk. n = 3. Scale bar, 100 μm. Schematic diagram of the procedure of establishing ciPSC lines. ciPSCs express key pluripotent markers Oct4, Sox2 and Nanog. Scale bar, 100 μm. ciPSCs have developmental potentials to form teratomas containing tissues from all three germ layers. Scale bar, 100 μm. Chimeric mice were generated from ciPSCs.
Figure 1A. Relative antiviral activity of IFNα (circles) or IFNλ (triangles). AEC cultures were stimulated for 4hrs with stated IFN at specified concentrations (ng/ml) and induction of indicated ISGs was assessed by qPCR (data shown is representative of four independent experiments, n=3-4). B, C. Mice were pretreated with equivalent doses of IFNα (1.45μg/50μl) or IFNλ (2.6μg/50μl) or Veh Ctrl (squares, 50μl PBS) 24hrs prior to infection with PR8; weight loss and survival was assessed throughout infection (B), and viral load (C) assessed at 4dpi (data shown is representative of two independent experiments, n=8-10 (B), n=3 (C)). D, E. Mice were infected with PR8 and treated with equivalent doses of IFNα or IFNλ or Veh Ctrl at days 2, 4 and 5 post infection; survival and weight loss was monitored (D, data pooled from 4 independent experiments, n=12-29) (data representative of two independent experiments, n=3-5). D, E. Mice were infected with PR8 and treated with equivalent doses of IFNα or IFNλ or Veh Ctrl at days 2, 4 and 5 post infection; viral load assessed at 4dpi (E) (data representative of two independent experiments, n=3-5).
Figure 2In vitro exflagellation rate of male gametocytes indicating male gamete formation. mean ± SEM from three experiments. Western blot of P28 protein expression in the non-activated gametocyte (NAG) and activated gametocyte (AG) of 17XNL and Δap2-o3 strains. BiP as loading control. The numbers are the relative intensities of P28 band normalized to the BiP band in the blot. Co-staining of P28 and GAP45 (IMC protein) in female gametes (P28+ only) and zygotes (P28+/GAP45+) of the 17XNL, Δap2-o3 and Δcdpk4 strains. Percentages of female gametes and zygotes were indicated respectively. n is the number of cells counted. Scale bars = 5 μm. Note: the signal of P28 and GAP45 is reduced similarly in all Δap2-o3 female gametes and zygotes compared to that of 17XNL. Time-course analysis of zygote to ookinete development in vitro. Upper diagrams indicate morphological changes from zygote to ookinete. Quantitative counting of different stages during parasite development in D. Day 7 midgut oocyst counts from mosquitoes infected with parasites, including 17XNL, Δap2-o3, Δmap2, or Δnek4 strain alone as well as mixtures of Δmap2/Δnek4, Δap2-o3/Δmap2, and Δap2-o3/Δnek4. Δnek4 and Δmap2 are female and male gamete-defect parasites, respectively. n is the number of mosquitoes dissected, Mann-Whitney test applied, two experiments repeated. Schematic of the defects caused by AP2-O3 deficiency during female gametocyte to ookinete development.
Figure 5A. Representative immunoblots from GluA2 coIP probed for Sec23, Sec13, and GluA2 after no DHPG, 10, 30, and 60 minutes of treatment with DHPG. B. Graph depicting the kinetics of the association between GluA2 and Sec23 as well as GluA2 and Sec13 after treatment with DHPG for the same points in time as in A. For Sec23, 10 min DHPG, n = 16; 30 min DHPG, n = 15; 60 min DHPG, n = 7, p < 0.0001, ANOVA. For Sec13, 10 min DHPG, n = 7; 30 min DHPG, n = 14; 60 min DHPG, n = 6, p < 0.01, ANOVA. C. Representative images of total expression levels of Sec23 (top) and Sec13 (bottom). Expression levels do not change in the presence of DHPG. All error bars represent s.e.m.
Molecular structural characteristics of the Ctf19-Mcm21 binding motif of Okp1.Figure 6A) Plots showing deuterium-exchanged peptides, after deuterium exchange of 10 sec of Okp1 in Ctf19D-RWD-Mcm21D-RWD-Okp1229-336, in full-length COMA, or in Ame11-260-Okp1123-336. Plots for full time course are in Appendix Fig S6C. B) Deuterium-exchange of peptides, from deuterium exchange data after 1200 sec of deteurium-exchange of Ctf19 and Mcm21, mapped onto the surface of the structure of the Ctf19-Mcm21 D-RWD domains (PDB code: 3ZXU); left: Ctf19-Mcm21 in COMA; right: Ctf19-Mcm21 alone. Residues without deuterium-exchange data are coloured dark grey. Plots for full time courses are in Appendix Fig S6D,E. C) Semi-transparent surface representation of our Ctf19D-RWD-Mcm21D-RWD-Okp1295-360 crystal structure, with secondary structure cartoon-diagram underneath; left: in a similar orientation as Ctf19-Mcm21 in B. D) Close-up view of the Okp1 binding site of Ctf19-Mcm21 with Ctf19-Mcm21 binding motif of Okp1; Okp1 residues, and some Ctf19 or Mcm21 sidechains, are shown as sticks (nitrogen: blue, oxygen: red); Ctf19-Mcm21 shown as secondary structure cartoon-diagram. E) Sequence alignment of budding yeasts' Ctf19-Mcm21 binding motif of Okp1. Similar residues are coloured red; conserved residues are on red background. We show full alignment in Fig EV5. F) Representative baseline-subtracted raw isothermal calorimetry data and derived binding isotherm from titration of Okp1-derived peptide, with the Ctf19-Mcm21 binding motif, to full-length Ctf19-Mcm21. Mean value for dissociation constant (Kd) and its standard error of regression are from three independent experiments. G) Plots from microscale-thermophoresis measurements with fluorescently labeled full-length Ctf19-Mcm21 and with Okp1-derived synthetic peptide (same as in F), or with Okp1-peptide variants that differ in one or two residue positions from the native Okp1 sequence (e.g. N330A: N330A). Normalized fluorescence, from which the fluorescence of unbound Ctf19-Mcm21 had been subtracted (ΔFnorm), is plotted against peptide concentration (logarithmic scale). For dissociation constants (Kd), we calculated mean value and confidence interval (68 % probability that Kd is within given range) with data from three independent experiments. Variant N330A-K332P had no detectable binding in the peptide concentration-range that we measured.
Figure 5(A) MCF10A cells were lysed and anti-TRIP6 or control (IgG) antibodies were used to isolate immune complexes. Immune complexes and lysates were probed by western blotting for vinculin and TRIP6. (B) MCF10A cells were either not treated (control) or treated separately by growth to high density, serum starvation, Latrunculin B, Blebbistatin, EGTA, or Y27632 treatment, and stained using anti-vinculin antibodies by immunofluorescence. Scale bar=20µm. (C) MCF10A cells grown at high density on PDMS membranes and were stretched (or not) at 17% elongation for 2 hours and fixed while under tension and stained for vinculin. Scale bar=20µm. (D) Vinculin was knocked down using two different siRNAs or control siRNA in MCF10A cells and the cell lysates were probed by western blotting for phospho-LATS1 (T1079 and S909), phospho-YAP (S127), LATS1, YAP, vinculin, and tubulin antibodies and the relative levels quantified (Mean ± SD; n=3; **P≤0.01, T-test). (E) Vinculin was knocked down as described in (D) and cells were stained for YAP and vinculin. Merged image shows YAP (green), vinculin (red), and DNA (blue). Quantification of at least 100 cells is shown (Mean ± SD; n=3; ***P≤0.001, Fisher's test). Scale bar=20µm. (F) Vinculin was knocked down as described in (D) and the levels of vinculin and YAP target gene expression was analyzed using RT-qPCR (Mean ± SD; n=3; ***P≤0.001, ****P≤0.0001, T-test). (G) Vinculin was knocked down in MCF10A cells as described in (D), grown at high density on PDMS membranes and cells were stretched (or not) at 17% elongation for 2 hours and the levels of vinculin and YAP target gene expression was analyzed using RT-qPCR (Mean ± SD; n=3; ***P≤0.001, ****P≤0.0001, T-test).
Complementary oscillations of glucose versus glutamine-derived fluxes in TCA cycle.Figure 5Figure 5: Complementary oscillations of glucose versus glutamine-derived fluxes in TCA cycle. (a-i) Oscillations in metabolic flux throughout the cell cycle (in mM/h), computed based on metabolic modelling of measured oscillations in metabolite concentrations and isotopic labeling (red and green marks represent optimal estimates of transient flux with 95% c.i.). Blue lines represent average fluxes inferred in a non-synchronized cell population. As shown, glucose-derived flux into TCA cycle peak in late G1 phase, while oxidative and reductive glutamine metabolism dominates S phase.
Figure 1(A) The bar graph represents top 10 biological pathways upregulated in gene ontology (GO) based Reactome pathway analysis using a set of genes induced (1.5 fold, p<0.05 (Wald Test), 3 biological replicates) in RNA-seq analysis in IRGM shRNA knockdown HT29 cells compared to control shRNA cells. Heatmaps were generated for sentinel interferon-regulated genes (three biological replicates) using 'ComplexHeatmap' library using 'R' Bioconductor package where the gene expression matrix was transformed into z-score. The heat map was generated from the common genes present in the three GO terms indicated by the three black lines. The numbers on the bars indicate the p-value of that particular GO term. (B) Interferome database analysis with a set of genes induced (1.5 fold, p<0.05 (Wald Test), 3 biological replicates) in IRGM shRNA knockdown HT29 cells compared to control shRNA cells. The Venn diagram depicts the total number of upregulated type I and type II IFN-regulated genes in IRGM KD cells. (C) Network pathway analysis using IPA. The molecular network of genes connected with the top 4 functions associated genes (1.5 fold, p<0.05 Wald Test, 3 biological replicates) upregulated in IRGM knockdown HT29 cells. (D) The qRT-PCR validation of RNA- seq data in control and IRGM KD HT29 cells. (E) The bar graph represents the top pathways upregulated in GO-based Reactome pathway analysis using set of genes induced (1.5 fold, p<0.05 (Wald Test), 3 mice each group) in the brain of Irgm1-/- mice compared to Irgm1+/+ wild type mice. Heatmaps were generated for sentinel interferon-regulated genes (three biological replicates) using 'ComplexHeatmap' library using 'R' Bioconductor package where the gene expression matrix was transformed into z-score. The heat map was generated from the common genes present in the three GO terms indicated by the three black lines. The numbers on the bars indicate the p-value of that particular GO term. (F) Interferome database analysis with a set of genes induced (1.5 fold, p<0.05, 3 biological replicates) in the brain of Irgm1-/- mice compared to Irgm1+/+ wild type mice. The Venn diagram depicts the total number of upregulated type I and type 2 IFN-regulated genes in Irgm1-/- mouse brain. (G) Network pathway analysis using IPA. The molecular network of genes connected with the top 5 functions/diseases associated with genes upregulated in Irgm1 knockout mouse brain. (H) The qRT-PCR validation of RNA- seq data in Irgm1+/+ and Irgm1-/- mouse brain.
Figure 6(A) Representative images of conventional TEM analysis of RPE of WT, DMSO-treated miR-211-/- mice and NSC668394-treated miR-211-/- three-month-old mice. NSC668394-treated miR-211-/- mice show rescue of accumulation of double-membrane phagolysosome-like structures containing poorly processed POS (red arrows) compared to DMSO-treated miR-211-/- mice. Scale bar 1 mm. Data information: (OS) outer segment (RPE) Retinal Pigment Epithelium. (B) Confocal images of representative eye cryosections immunostained with anti-Rhodopsin (green) antibody from WT and miR-211-/- mice at three months of age after DMSO or NSC668394 treatment, which are sacrificed 3h after light on at 10 AM (diurnal condition). Nuclei are counterstained with DAPI (blue). At least n = 6 mice per group. Scale bar 100 μm. The enlarged box highlights rhodopsin accumulation in the RPE (green spots). Scale bar 5 μm. Autofluorescence from lipofuscin granules from WT and miR-211-/- mice at three months of age after DMSO or NSC668394 treatment. Representative images of retina cryosections immunostained with anti-Cone Arrestin (green) antibody from WT and miR-211-/- mice at three months of age after DMSO or NSC668394 treatment. Nuclei are counterstained with DAPI (blue). N = at least 6 mice per group. Scale bar 100 μm. Data information: (OS) outer segment; (ONL) outer nuclear layer; (RPE) Retinal Pigment Epithelium. (C) Graphs showing number of lipofuscin granules from the RPE of mice as in (B). Bar graphs represent mean values ± s.e.m. Mann and Whitney test (miR-211-/- DMSO vs WT and miR-211-/- NSC668394 vs DMSO treated mice) ***p ≤ 0.005. *p ≤ 0.05 (n=6 mice). (D) Graphs show cone percentage (cones/area) from the retina of mice treated as in (B). Error bars represent s.e.m. Mann and Whitney test (DMSO vs WT and NSC668394 vs DMSO treated mice) ***p ≤ 0.005 (n=6 mice). (E) Representative flicker traces at three months of age show the rescue of flicker responses of NSC668394-treated miR-211-/- mice (green lines) compared to DMSO-treated miR-211-/- control mice (red lines). WT mice were used as a control (black lines). Flicker recordings were performed with light intensities ranging from 10−4 to 15 cd s/m2 in steps of 0.6 logarithmic units at 6 Hz frequency. (F) Flicker responses, plotted as a function of stimulus intensity, from WT (black lines), DMSO-treated miR-211-/- (red lines) and NSC668394-treated miR-211-/- (green lines) mice, at three months of age. The amplitude of the recordings from NSC668394 miR-211-/- treated mice is significantly rescued compared to DMSO-treated miR-211-/- mice. WT mice were used as a control. Error bars represent s.e.m. ANOVA test (DMSO vs WT and NSC668394 vs DMSO treated mice) **p ≤ 0.01, ***p ≤ 0.005.
Figure 2Effects of CLIP170 on fusion of viral particles by endocytosis. CHME3 cells either treated with control or CLIP170 siRNAs (A) were infected with Mock or HIV-1-VSV-BlaM-Vpr. Representative FACS analysis of cells (n = 3) showing ∼ 9% (Control siRNA) and 3% (CLIP170 siRNA) shift (A) from green (uncleaved CCF2) to blue (cleaved CCF2). % infected cells is indicated at the top left corner of each plot and quantification from independent repeats is presented as bar graphs at the right side of each FACS plot. HIV-1-VSV-luc infection is enhanced in Flag-CLIP170, but not in Flag control, expressing CHME3 cells. Molecular weight markers (in kDa) are shown to the right of WBs in C. Effects of CLIP170 on fusion of viral particles by endocytosis. CHME3 cells expressing Flag control or Flag-CLIP170 (D) were infected with Mock or HIV-1-VSV-BlaM-Vpr. Representative FACS analysis of cells (n = 3) showing no difference (D) from green (uncleaved CCF2) to blue (cleaved CCF2). % infected cells is indicated at the top left corner of each plot and quantification from independent repeats is presented as bar graphs at the right side of each FACS plot. (E) The percentage fused double-labelled (GFP-Vpr+/S15-tomato-) HIV-1-DL-WT are not affected in CHME3 cells depleted of CLIP170. Data represent mean of ≥895 virus particles analyzed in ≥11 fields of view (≥17 cells) for each condition. One-way ANOVA was used to calculate statistical significance (mean ±SEM). (F) Percentage HIV-1-WT-GFP-Vpr particles within 2 μm of the nucleus is reduced in CHME3 depleted of CLIP170 at 2 and 4 h.p.i, scatterplot with mean of ≥354 virus particles quantified in ≥13 cells per sample. Statistical significance was determined by one-way ANOVA * = p<0.05, *** = p<0.001. Infected cells in F analyzed by live cell imaging. Scatterplots representing displacement over time (velocity) Each dot represents one viral particle tracked, red line indicates the mean. 188 trajectories were analyzed from 25 movies. Statistical significance was determined by t test. *** = p<0.001. Infected cells in F analyzed by live cell imaging. cumulative distance travelled over time (speed) (H). Each dot represents one viral particle tracked, red line indicates the mean. 188 trajectories were analyzed from 25 movies. Statistical significance was determined by t test. *** = p<0.001. (I) The induction of Ac-MTs is blocked in HIV-1-VSV-luc (Virus) infected, but not in uninfected (NI) or Mock infected CHME3 depleted of EB1, but not CLIP170 at 6 h.p.i. Tyrosinated MTs (Tyr-MTs) are not affected. Representative fields are shown. Similar results were obtained in three independent experiments. Scale bar, 50 μm.
SARS-CoV-2 nucleocapsid protein undergoes LLPS at physiological conditions.Figure 3A) Phase separation over time as monitored by turbidity of 50 μM MBP-N after addition of TEV protease in the presence of salt (183 mM NaCl) and 0.3 mg/ml RNA at pH 7.4 (50 mM Tris), pH 6.1, pH 5.5, or pH 4.9 (pH 6.1 and below in 20 mM MES). Error bars represent standard deviation of three replicates. For samples at B) pH 7.4 or C) pH 5.5, DIC micrographs of 50 μM MBP-N in the presence of salt (183 mM NaCl) without or with TEV protease (to cleave MBP from N) and without or with 0.3 mg/mL desalted total torula yeast RNA. Scale bars represent 50 μm.