EMBO/sd-character-level-ner huggingface dataset:
Figure 4(A) Representative images of control cells (upper panel) or cells treated with α-synucleinfibrilsATTO-550 (bottom panel) for 16 hours; in red: α-synucleinfibrils and in green: WGAAlexa-488 (plasma membrane dye marker), blue: DAPI. Scale bar represents 10 μm (n = 3 independent experiments).(B) Relative percentage of TNT-connected cells after α-synucleinfibrils treatment as in A shows an increase in TNT number upon fibrils treatment. Data are mean ± s.e.m (***, p < 0.001 by paired, two-tailed Student's t-test). (C) Confocal images showing one TNT connecting two cells loaded with α-synucleinfibrilsATTO-550 (in red) 16 hours after co-culture and stained with WGAAlexa-488 (in blue). The image represents a Z-projection of the several middle stacks where the TNT is located (top panel). Insets show 3D reconstructions of the TNT using ICY software (middle panel: merge; bottom panel: red channel). Several α-synucleinfibrils (red) are present in the lumen of the TNT (WGAAlexa-488 in white). Scale bars represent 10 μm (confocal images) and 4 μm (insets). (D) Confocal image showing TNT connecting a donor cell containing α-synucleinfibrils and GFP transfected acceptor cell (top panel) strongly suggesting α-synucleinfibrils transfer from donor to acceptor cell. Insets show 3D reconstructions of TNT containing α-synucleinfibrils in the lumen of the TNT; in red: α-synucleinfibrils, in green: GFP transfected acceptor cells in blue: WGAAlexa-488. Scale bars represent 10 μm and 3 μm for the insets. (E) Relative percentage of TNTs in cells containing α-synucleinfibrils untransfected (control) or transfected with Myosin 10 (Myosin 10). Overexpression of Myosin 10 in the donor cell population led to an increase in the number of TNTs. (F) Quantification of the average number of ATTO-550α-synucleinfibrils per acceptor cell after 24 hours co-culture with donor cells containing α-synucleinfibrils untransfected (control) or transfected with Myosin 10 (Myosin 10). Note that Myosin 10 overexpression increases both the transfer of DiIvesicles and the number of transferred α-synuclein puncta in acceptor cells; n = 3 independent experiments (**, p < 0.01 by two-tailed Mann Whitney test). (G) Quantification of the relative proportion of TNTs in co-culture of donor cells containing α-synuclein puncta and GFP acceptor cells under sub-confluent or sparse culture conditions. Data are mean ± s.e.m; n = 3 independent experiments (*, p < 0.05 by paired, two-tailed Student's t-test).(H) Quantification of the percentage of acceptor cells containing α-synuclein puncta of the co-culture in G. n = 3 independent experiments (*, p < 0.05 by two-tailed Mann Whitney test). Condition impairing TNT formation (i.e., sparse condition) induces a drastic decrease of TNT number and α-synucleinfibrils transfer compared to condition allowing TNT formation (sub-confluent condition).
Figure 3Effect of ASO-mediated inhibition of 5'-tsRNAs on alkaline phosphatase activity in RA-treated mESCs. (n = 3), Error bars represent SEM, significance was calculated using unpaired t-test. Relative expression of stemness markers in RA-induced differentiation mESCs blocked for 5'-tsRNA function with ASOs. (n = 2) Error bars represent SEM, significance was calculated using unpaired t-test. Volcano plot of the transcriptome depicting the overexpressed transcripts (in green) and downregulated transcripts (in pink) in ASO treated-differentiating cells. KEGG pathway analysis of the transcripts upregulated in a RA-mediated differentiating mESCs blocked for tsRNA function. QPCR validation of transcripts involved in the stem cell signalling pathway that are upregulated upon 5'-tsRNA knockdowns using ASOs. (n =2) Error bars represent SEM, significance was calculated using unpaired t-test.
a,b) Analysis of FL83B mouse liver cells pretreated with mouse recombinant IL-1β (2 ng/ml) (a) or TNF (2 ng/ml) (b) for 24 h, then stimulated with insulin (200 nM) for 10 min.figf6(a,b) Analysis of FL83B mouse liver cells pretreated with mouse recombinant IL-1β (2 ng/ml) (a) or TNF (2 ng/ml) (b) for 24 h, then stimulated with insulin (200 nM) for 10 min. p-Akt (Ser473) was determined by flow cytometry (right) with quantification shown in the graph at left (a). p-Akt (Ser473) was determined by immunoblotting (b). (c) p-IRS1 (Ser307), determined by immunoblotting, in FL83B mouse liver cells treated with either IL-1β or TNF for 24 h. (d-f) Akt Ser473 phosphorylation measured by flow cytometry (d,e) or immunoblotting (f). FL83B cells (d,e) or WT primary hepatocytes (f) were pretreated with conditioned medium (CM) generated from WT, Pycard−/− (d), Nlrp3−/− or Casp1−/− (e) macrophages for 24 h, then stimulated with insulin. Control medium was obtained from wild-type BMM cultures without stimulation. (d-f) Akt Ser473 phosphorylation measured by flow cytometry (d,e) or immunoblotting (f). FL83B cells (d,e) or WT primary hepatocytes (f) were pretreated with conditioned medium (CM) generated from WT, Pycard−/− (d), Nlrp3−/− or Casp1−/− (e) macrophages for 24 h, then stimulated with insulin. Control medium was obtained from wild-type BMM cultures without stimulation (g) Akt serine 473 phosphorylation measured by flow cytometry. FL83B cells were pretreated with WT or Pycard−/− CM in the absence or presence of the IL-1 receptor antagonist anakinra (1 μg/ml) for 24 h, then stimulated with insulin. (h) p-Akt (Ser473) analyzed by immunoblotting and quantified by densitometric analysis in FL83B cells pretreated with WT CM in the absence or presence of either anakinra or neutralizing antibody to TNF. The results shown are representative of three independent experiments and are expressed as mean ± s.d.
Figure 6A. In vitro transcribed mt-tRNAMet was methylated at C34 using recombinant NSUN3 and 3H-labelled S-adenosyl methionine as a methyl group donor. Radiolabelled mt-tRNAMet was re-extracted and then subjected to oxidation assays without protein (-), with maltose binding protein (MBP), with the dioxygenase FTO or using wildtype (ABH1) or mutant (R338A, D233A) His14-MBP-ABH1. Besides ABH1 controls lacking a-ketoglutarate (KG) or Fe2+ ions, all samples contained a-ketoglutarate and Fe2+ ions. After oxidation, RNA was precipitated and tritium released upon oxidation of radiolabelled mt-tRNAMet was quantified in the supernatant and counts per minute (CPM) are shown for experiments performed in triplicate with error bars indicating ± SD (upper panel). Pelleted RNA was separated on a denaturing polyacrylamide gel and exposed to an X-ray film to analyse the tritium retained (3H-Me). B. Synthetic anticodon stem loop (ASL) was radioactively labelled and subjected to oxidation assays that were performed and analysed as described in (A) using no protein (-), MBP or wildtype His14-MBP-ABH1 (ABH1). Experiments were performed in triplicate with error bars indicating ± SD. C. Anion exchange HPLC analysis was performed on synthetic m5C containing ASL (20nt) before and after oxidation by ABH1. The small shift in retention time indicates formation of f5C modified RNA. The ABH1 oxidation product was then treated with NaBH4 to generate hm5C-modified RNA. All three samples were analysed by ESI-MS and the molecular weight (m.w.) is indicated on the HPLC trace. Only the f5C-containing RNA was labelled efficiently with 1-ethyl-2,3,3-trimethylindoleninium-5-sulfonate (TMI). D. The different retention times of m5C, hm5C and f5C-modified ASL RNA were confirmed by co-injection of samples shown in (C). HPLC was performed as in C.
Figure 2A Box plots of the Em/Ad expression ratios of GATA6 (left) and NKX2-1 (right) in EBCs from controls (Ctrl, n=65) or lung cancer (LC, n=48) patients (training set). Each point represents one sample. B Learning of the SVM on the training set of EBCs. Left, the log2-transformed Em/Ad expression ratios of GATA6 (x-axis) and NKX2-1 (y-axis) detected in the EBCs of the training set were plotted. The SVM combined the Em/Ad of GATA6 and NKX2-1 of each sample and created a linear classifier. The solid line represents the decision boundary of this classifier, which reliably separates control (green dots) and LC (red dots) samples. Filled circles, correctly classified samples; open circles, wrongly classified samples. Right, box plot of the LC score on the training set of EBCs. C External validation of the LC score. Left, the log2-trasnformed Em/Ad expression ratios of GATA6 and NKX2-1 detected in an independent set of blinded collected EBCs were plotted as in (B). The SVM based classifier confirmed its discriminatory power between control and LC samples. Right, box plot of the LC score on the validation set of EBCs. D ROC analysis confirmed the increase in performance of the LC score based classification on the validation set of EBCs (black line) over a classification relying only on Em/Ad expression ratios of GATA6 (pink line) or NKX2-1 (blue line). The orange diamond represents the optimal operating point of the SVM classifier, i.e. the point on the curve with maximal Youden´s J index. E Performance of the LC score on the validation set of EBCs. PPV, prospective predictive value; NPV, negative predictive value; TPR, true positive rate or sensitivity; TNR, true negative rate or specificity.
Epigenomic profiling using a single tissue section.Figure 1(A) Schematic diagram of the ChIL protocol for tissue. (B-D) Immunofluorescent images of mouse liver (B), testis (C) and heart (D). H3K27ac, PolII-S5P, and laminin were stained with specific primary antibodies and visualized using fluorescent labeled anti-mouse ChIL-probe (red: H3K27ac and PolIIS5P) and anti-rabbit IgG (green: laminin). DNA was counterstained with Hoechst 33342. Scale Bar: 20 µm (left images), 10 µm (right images). (E) Genome browser images of ChIL-seq for H3K27ac and PolII-S5P and bulk tissue RNA-seq data at the Alb locus in liver tissues. (F) Library complexity of ChIL data. Poisson represents an ideal case of the uniform probability of obtaining reads from the mouse genome, whereas preseq refers to the future/past predictions of a species discovery curve of sequenced reads using Preseq(Daley & Smith, 2013a). Black circle indicates the read number we sequenced for this prediction. (G) Breakdown of mapped reads at the annotated genomic regions. Gene body: 3'-UTR, exon, intron, 5'-UTR; Others: ncRNA, miRNA, snoRNA, and pseudogenes. The proportions of the annotated region on the mouse genome are shown as "Genome" at the bottom lane. (H) Genome-wide correlation at 10 kbp bins. Hierarchical clustering of Pearson's correlation coefficient of log-transformed ChIL-seq counts is shown.
Human HOIP is ubiquitinated at K784 and regulates NF-κB.Figure 1. A scheme of procedures for the UbiCRest-based assays employed to analyze HOIP-modification with ubiquitin chains. Total cell extracts from HEK293T cells transiently expressing Myc-HOIP and Myc-OTULIN C129A, a catalytic inactive mutant, subjected to GST-linear-TUBE pulldown followed by UbiCRest using recombinant deubiquitinases (vOTU, OTULIN and USP21). . UbiCRest assays to evaluate ubiquitin chain types on HOIP examined by immunoblotting. Immunoblotting of samples using antibodies as indicated. Ponceau S staining used for monitoring GST-liner-TUBE input. Representative data shown from three independent experiments. . Mass spectrometry spectra corresponding to a peptide containing HOIP-K784 with double Gly (114+K). . Domains of human HOIP and identified ubiquitination sites at K454, K458, K735 and K784. . Multiple sequence alignment of different HOIP orthologues illustrating the position K735 and K784 according to the ClustalX colour scheme. Sequences were retrieved from the NCBI protein database with the following accessions: Homo sapiens (NP_060469.4), Canis lupus (XP_005623312.1), Mus musculus (NP_919327.2), Monodelphis domestica (XP_007479924.1), Xenopus laevis (NP_001090429.1), Alligator mississippiensis (XP_006259801.1), Takifugu rubripes (XP_003968217.2) and Drosophila melanogaster (NP_723214.2). . Co-immunoprecipitation analysis of the HOIP K784R mutant with SHARPIN and HOIL-1L using total cell extracts of HEK293T cells transiently expressing Myc-HOIP wildtype (WT), or Myc-HOIP-K784R with HOIL-1L-HA and Flag-SHARPIN. Anti-Vinculin antibody used to monitor protein loading. Representative data shown from three independent experiments. . Luciferase-based NF-κB gene reporter assays using Myc -HOIP wildtype (WT), a catalytic inactive mutant C885A, ubiquitination site mutants of K454R, K458R, K735R, K784R co-transfected with HOIL-1L-HA and Flag-SHARPIN. Luciferase signal was normalized to an internal Renilla control signal.
Figure 1a) Photomicrographs represent 2D optical sections from a Z stack acquisition of whole mounted skin displaying IFE harbouring multiple clones of different colours at 3, 5, 12 and 24 weeks (bar represents 50 μm). In each image a clone is circled in white, illustrating the progression of clone size at different times. b) Histogram charts showing the relative frequency of clones according to their size between 3 and 24 weeks in mice induced at P21 and P42. The frequency of larger clones increases over time. Chi square test p<0.0001 for P21 group and p=0.0056 for P42 group. The number of clones evaluated at each time point is indicated above each histogram series. c) and d) Correction for contiguous clones harbouring a same colour. c) Snapshots from a computer simulation on a 2D lattice attributing 6 colours to basal keratinocytes. The Fig illustrates the typical evolution of clone clusters in a 2D lattice simulating the basal layer with six different colours at different time points. The real clone size is simulated by attributing an infinite number of colours to the lattice and ensuring that each basal cell is uniquely labeled. d) Plots show the probability for an individual keratinocyte to belong to a clone of a given size. Experimental data are represented in green bars. Red curve represents the 5-colour simulation and the blue curve the real size simulation. Although the frequency of large clones is overestimated in experimental data due to the limited 5-color palette, it is apparent that real clone size progresses over time. e) Histogram charts showing the distribution of clones at D1-3 following tamoxifen induction according to total number of cells and number of basal cells. f) Histogram charts showing the relative frequency of clones according to their size between 3 and 5 weeks in groups injected at P21 and P42 with a single low dose of Tamoxifen to perform a low density staining. The frequency of larger clones increases over time. Chi square test p=0.0013 for P21 group and p=0.0296 for P42 group. All data are represented as mean ± s.e.m.
Scheme of complex-centric proteome profiling by SEC-SWATH-MS.Figure 1Workflow to quantify cellular complexes in five steps, extending the targeted analysis concept from peptide-centric interpretation of SWATH-MS data to the levels of protein and protein complex detection from size exclusion chromatographic fractions Specific steps of targeted, complex-centric analysis of co-fractionation data in the CCprofiler package.
Anaphase centrosomal actin aster formation depends on the Arp2/3 activator WASH1 A.Figure 6Time-lapse of HeLa cells expressing GFP-alpha tubulin and RFP-lifeact treated with control siRNA or siRNA against WASH1 for 48 hours arrested at prometaphase and forced to exit mitosis with Ro3306 addition (t=0). Scale bar = 5µm. Quantification of actin around the centrosome in (A), showing the failure to accumulate actin around the centrosome in siWASH. N = 4 experiments. Error bars represent standard deviation. Western blots of lysates from cells treated with siRNA control and siRNA against WASH1 probed with WASH1 antibody, showing the reduction in WASH levels following treatment of cells with siRNA against WASH. Scale bar = 5µm and 2µm in zoom. Z-projection of HeLa cells expressing GFP-centrin1 treated with control siRNA or siRNA against WASH, arrested at prometaphase or forced to exit mitosis and immunostained with phalloidin. Scale bar-5µm Quantification of actin around the centrosome in (F), showing the failure to accumulated actin around the centrosome during forced exit in cells treated with siRNA against WASH. siControl-STLC= 1 ± 0.0189, n=165, siWASH-STLC=0.9138 ± 0.0298, n=118, siControl-STLC+RO-3306=1.911 ± 0.06221, n=147, siWASH-STLC+RO-3306=1.103 ± 0.0301, n=140. Error bars represent standard deviation. P<0.0001, one-way ANOVA. Time-lapse from in vitro assay with centrosomes isolated from cells undergoing forced exit were pre-treated with either no antibody or anti-WASH1 antibody for 1 hour. Scale bar- 10µm Quantification of in vitro assay from (F), showing the reduction in actin nucleation around centrosomes when they are pre-treated with anti-WASH1. Error bars represent standard deviation. 2 independent experiments.
Features of PC Loss in npc1 Mice.Figure 2(A) Sections from the cerebellar vermis of a 70-d-old npc1−/− mouse were stained with anti-Calbindin (green) to visualize PCs and 7AAD (red) for nuclei. Although there are scant PCs remaining in lobule II, the PCs in lobule X have not decreased. (B) Quantification of progressive anterior-to-posterior PC loss. PC densities from 30-d npc1+/+ and 30-, 50-, and 70-d npc1−/− mice were quantified from five sections from two mice each. Error bars show standard deviation. (C) Bergmann glia visualized in 70-d npc1+/+ and npc1−/− mice using anti-S100β (green) have normal morphology. Note that the glial cell bodies near the PC layer and the radial processes extending into the molecular layer are intact in the npc1−/− mouse despite the loss of PCs. (D) Oligodendrocyte cell bodies stained with anti-CC1 (green) have a similar distribution and morphology in the cerebellarwhite matter of 70-d npc1−/− and wild-type mice. TOTO-3 (red) was used as a nuclear counterstain. Bar = 5 μm. (E) Axonal spheroids in npc1−/− mice. Spheroids (arrows), seen as Calbindin-positive (red) swellings surrounded by myelin (anti-myelin basic protein; green), are numerous in the npc1−/− cerebellum (50-d-old, lobule X shown) but are not seen in the wild-type control. Bar = 5 μm. (F) Sections from wild-type and npc1 mice were stained with filipin to visualize free cholesterol. PCs from npc1 mice show an accumulation of intracellular cholesterol at 30 d regardless of their lobular location. Images are oriented such that the PCs (arrowheads) are in the center of the field, with the molecular layer above and the inner granule cell layer below.
Figure 3Heatmap showing gene expression profile of HEKn silenced by either SCR or si-uc.291 (2) siRNAs by gene microarray analysis; n=4 technical replicates using 1 human donor; p<0.05, Student's t-test, abs (FC)>2. RT-qPCR confirming the efficiency of silencing. Data shown are mean ± s.d; the same samples of (A) were used; **p<0.01, Student's t-test. Volcano-plot showing fold changes of differently expressed genes from (A): up-regulated genes are shown in red and down-regulated genes are shown in grey. GO terms analysis for genes down-regulated upon uc.291 depletion from (A). GSEA analysis comparing a query gene set of modulated genes from (A) with GO biological process signatures associated with epidermis development and keratinocyte differentiation. NES=normalised enrichment score, FDR=false discovery rate. Kolmogorov-Smirnov statistic was used. Heatmap showing gene expression of the most down-regulated genes located within epidermal differential complex (EDC) upon depletion of uc.291 from (A). Scheme of the Epidermal Differentiation Complex (EDC) genes. Validation of array data by RT-qPCR showing mRNA quantification of thirteen EDC genes down-regulated upon si-uc.291. Validation of array data by RT-qPCR showing mRNA quantification of eight EDC genes down-regulated in uc.291-depleted organotypic human epidermis; n=2 technical (#1 and #2) from 1 human donor.
EGFR kinase activity is required for IRF3 activation.Figure 3(A) EGFR was not required for ligand-induced STING dimerization: MEFs were transfected with HA-tagged STING and treated with gefitinib or DMSO before cGAMP transfection, 4h later STING was immunoprecipitated with anti-HA antibodies and analyzed by Western blot. (B) EGFR was required for the activation of IRF3, but not TBK1: MEFs were pretreated with gefitinib for 1h, then treated with cGAMP for 2h; cell lysate were subjected to Western blot analysis using the indicated antibodies. (C) Nuclear translocation of IRF3, but not NFκB p65, required EGFR: MEFs were pre-treated with gefitinib (+) or DMSO (-) for 1 hour before transfecting cGAMP. Cells were harvested and nuclei were isolated at the indicated time points; total cell extracts (T) and nuclear extracts (N) were analyzed by Western blot. MEFs were pretreated with gefitinib for 1h, then treated with cGAMP for 2h; (D) IRF3, but not TBK1, binding to STING required EGFR cell lysates were immunoprecipitated with anti-STING antibody and analyzed by Western blot. (E) STING-IRF3 interaction in human cells required EGFR: WT and EGFR-/- (KO) HeLa cells were transfected with cGAMP, 2h later cells were lysed, immunoprecipitated with anti-STING antibody and analyzed by Western blot. (F) STING and IRF3 co-localized in the endosomes of stimulated cells: STING-/- MEF cells were transfected with STING-HA for 24 hours then treated with cGAMP for 1h. The cells were then stained with the HA (purple), CD63 (red) and IRF3 (green) antibodies. Imaging data were analyzed by the Fuji software to reveal co-localization as white dots. Scale bars represent 10 µ m. (G) EGFR was not required for ligand-induced phosphorylation of STING Ser366: EGFR-/- HeLa cells or gefitinib-treated and untreated WT HeLa cells were treated with cGAMP for the indicated time and cell lysates were analyzed by Western blot. (H) Kinetics of STING and IRF3 co-localization in the endosomes of stimulated cells: STING-/- MEF cells were transfected with STING-HA for 24 hours and treated with cGAMP for the indicated time. The cells were then stained with the HA (purple), CD63 (red) and IRF3 (green) antibodies and DAPI. Imaging data were analyzed by the Fuji software to reveal co-localization as white dots. Scale bars represent 10 µ m.