EMBO/SourceData huggingface dataset:
Figure 7One thousand ESCs of the indicated genotypes were placed into hanging-drop cultures to form embryoid bodies (EBs). After 3 days (referred to as day-0), differentiation was induced by exposure to differentiation medium (days-1 to days-3 after medium change). (A) Representative photographs of EBs of the indicated genotypes at day-0 and day-3 after differentiation induction (experiments were conducted in 3 biological replicates; n=5-42 EBs per biological replicate, scale bar: 0.02 mm).RNA-seq analysis was conducted on Tnfaip2-/- EBs vs. WT EBs on day-3 of differentiation induction of EBs (n=3 biological replicates). The heat maps depict the intersection of DEGs with the following gene sets (B) "stem cell maintenance: positive and negative regulators",RNA-seq analysis was conducted on Tnfaip2-/- EBs vs. WT EBs on day-3 of differentiation induction of EBs (n=3 biological replicates). The heat maps depict the intersection of DEGs with the following gene sets ;, (C) "mesoderm development"RNA-seq analysis was conducted on Tnfaip2-/- EBs vs. WT EBs on day-3 of differentiation induction of EBs (n=3 biological replicates). The heat maps depict the intersection of DEGs with the following gene (D) "ectoderm development" Tnfaip2 and 5 other stem cell-related genes (Tfcp2l1, Dppa3, Fbxo15, Zfp42 and Klf2) were additionally incorporated based on literature searches. The color scale represents the gene-wise z-score calculated from normalized gene expression levels. The asterisk refers to the gene identified in this study.(E) Representative image of immunofluorescence staining against the ectodermal marker (SOX1) and the mesodermal marker (T alias Brachyury) in WT and Tnfaip2-/- EBs on day-3 of differentiation induction. (3 repeat experiments were conducted on a total number of 9-10 EBs per genotype, scale bar: 20µm).(F) mRNA expression of Tnfaip2 measured by RT-qPCR in ESCs on the indicated days of differentiation induction of WT EBs [n=3 biological replicates; log-transformed data were normally distributed (P>0.05 as per Shapiro-Wilk test), the P value for the upregulation of Tnfaip2 on consecutive time points (day-0, -2 and -3) was calculated starting with the probability of maximum rank-based difference, i.e. having two groups of three data points perfectly separating, p1,b = 0.1 (bidirectional) and P1,u = 0.05 (unidirectional); the probability of finding a triple series of max. difference starting at some timepoint is Ptriple,tp = p1,b · p1,u · p1,u, and finding such a triple somewhere across the 4-step time series is Ptriple = Ptriple,tp + (1- p1,u) · Ptriple,tp = 0.0005.
Figure 8(B) The Volcano plots show the differentially expressed proteins identified from the proteome analysis of Tnfaip2-/- EBs versus WT EBs at the indicated days after induction of differentiation. Vim (marked in blue asterisk) was the only protein that featured among top 100 differentially expressed proteins at all time points by implementing selection criteria of an average log2ratio > +/-1.2 and a -log10Qvalue <2. Vim expression was reduced at all timepoints of in vitro differentiation in Tnfaip2-/- EBs vs. WT EBs (n=5 biological replicates, see also Dataset EV8 for top 100 regulated proteins). Sphere size illustrates the magnitude of log2fold changes as does color coding: blue-downregulated proteins, red-upregulated proteins; log2fold marking by color intensity: strong intensity >2, medium intensity > 1.5, faded color >1.(C) mRNA expression of Vim on indicated days of WT EB and Tnfaip2-/- EB differentiation (n=3 biological replicates; log-transformed data were normally distributed (P>0.05 as per Shapiro-Wilk test) and analyzed by multiple t-tests with Holm-Sidak correction for multiple testing).FACS analysis of lipid droplet (LD) content of WT (blue) and Tnfaip2-/- (red) EBs by BODIPY493/503 staining at the indicated days after differentiation induction: (D) Representative FACS blot of BODIPY staining. Unstained WT EBs served as a negative control (grey).FACS analysis of lipid droplet (LD) content of WT (blue) and Tnfaip2-/- (red) EBs by BODIPY493/503 staining at the indicated days after differentiation induction: (E) Quantification of staining intensity [n=3 independent cultures per genotype; data were normally distributed (P>0.05 as per Shapiro-Wilk test) and analyzed by multiple t-tests with Holm-Sidak correction for multiple comparisons].Transmission Electron Microscopy (TEM) was used to determine the number of LDs in EB cultures derived from WT and Tnfaip2-/- ES cells on day-1 after differentiation induction. (n=5 independent cultures per genotype, n=100 images per replicate). (F) Representative micrographs of TEM analysis of EB cells of the indicated genotype. Yellow arrows point to LDs, scale bars: 1 µm.Electron Microscopy (TEM) was used to determine the number of LDs in EB cultures derived from WT and Tnfaip2-/- ES cells on day-1 after differentiation induction. (n=5 independent cultures per genotype, n=100 images per replicate). (G) Quantification of LDs per TEM field. Data were not normally distributed (P<0.05 as per Shapiro-Wilk test) and analyzed by Mann-Whitney test.(H) UPLC-MS/MS analysis revealed a reduction in triacylglycerol (TAG) content in Tnfaip2-/- EBs compared to WT EBs at the indicated time points after differentiation induction [n=3 independent cultures of EBs per time point; data of EBs on day-0 to day-3 of differentiation were statistically analyzed; mean centered group data were normally distributed and (P>0.05 as per Shapiro-Wilk test) and analyzed by one-sided t-test with Holm Sidak correction for multiple testing].
Figure 9(A) Heatmap of CPT1A protein expression as measured in proteome analysis of Tnfaip2-/- and WT ESCs and during differentiation induction of EB cultures (n=5 biological replicates). The colour scale represents average log2ratios.(B) Expression of Cpt1a mRNA as determined by qRT-PCR in WT and Tnfaip2-/- EBs at the indicated days of differentiation induction [n=3 biological replicates; log-transformed data were normally distributed (P>0.05 as per Shapiro-Wilk test) and analyzed by multiple t-tests with Holm-Sidak correction for multiple comparisons].Differentiation-induced WT EBs and Tnfaip2-/- EBs were treated with palmitic acid (PA) and palmitoyl-L-carnitine (PC) (8μM each) or a vehicle control (C,D) The diameters of the rosette structure of differentiated WT EBs and Tnfaip2-/- EBs were measured on day-3 after differentiation induction. (n=3 repeat experiments with 7-62 EBs per experiment per group): (C) Representative photographs (scale bar: 0.02 mm) and (D) quantification of the diameter of the rosette structure of the indicated groups [data were not normally distributed (P<0.05 as per Shapiro-Wilk test) and thus analyzed by Mann-Whitney U test with Holm Sidak correction for multiple comparisons].RNA-seq was conducted on day-3 of differentiation induction of Tnfaip2-/- EBs that were either treated with PA/PC or with a vehicle control (n=3 independent pools of 7-62 EBs per group). The heat maps depict the intersection of DEGs with the following gene sets (E) "stem cell maintenance: positive and negative regulators",RNA-seq was conducted on day-3 of differentiation induction of Tnfaip2-/- EBs that were either treated with PA/PC or with a vehicle control (n=3 independent pools of 7-62 EBs per group). The heat maps depict the intersection of DEGs with the following gene sets ;, (F) "mesoderm development"RNA-seq was conducted on day-3 of differentiation induction of Tnfaip2-/- EBs that were either treated with PA/PC or with a vehicle control (n=3 independent pools of 7-62 EBs per group). The heat maps depict the intersection of DEGs with the following gene sets (G) "ectoderm development". Five other stem cell-related genes (Tfcp2l1, Dppa3, Fbxo15, Zfp42 and Klf2) were additionally incorporated based on literature searches. The asterisks indicate the reverted changes in gene expression that were seen in Tnfaip2-/- EBs versus WT EBs The color scale represents the gene-wise z-score calculated from normalized gene expression levels.(H) Representative image of immunofluorescence staining against ectodermal marker (SOX1) and the mesodermal marker (T alias Brachyury) on day-3 of differentiation induction of Tnfaip2-/- EBs +/- co-treatment with PA/PC. Three repeat experiments were conducted with a total number of 9 EBs per group
Figure 10Oct4-eGFP reporter MEFs or WT MEFs were infected with shRNAs Cells were co-infected with 4 reprogramming factors (OSKM) (A) Representative images of AP staining of iPSC coloniesOct4-eGFP reporter MEFs or WT MEFs were infected with shRNAs Cells were co-infected with 4 reprogramming factors (OSKM) and double-infected cells were FACS sorted and grown for 14 days, when reprogramming efficiencies were determined by FACS. (B) representative FACS profiles of Oct4-positive iPSCs on day 14, (C) quantification of the percentage of Oct4-GFP+ iPSCs in MEF cultures that were infected with 2 different shRNAs targeting Vim or a scrambled shRNA control [n=3 biological replicates per group; data were normally distributed (P>0.05 as per Shapiro-Wilk test) and analyzed by t-test with Holm-Sidak correction multiple comparisons].Oct4-eGFP reporter MEFs or WT MEFs were infected with combinations of shRNAs. (D) Representative FACS profiles of mouse-specific pluripotency cell surface marker SSEA1+ iPSCs on day 14 of reprogramming and (E) quantification of the percentage of SSEA1+ iPSCs in MEF cultures that were infected with the indicated shRNAs targeting Vim and/or Tnfaip2 [n=5 biological replicates; data were normally distributed (P>0.05 as per Shapiro-Wilk test) but showed unequal variance; data were analyzed by Brown-Forsythe and Welch ANOVA and Dunnett's multiple comparisons test].combinations of shRNAs. RT-qPCR was performed to determine (F) Vim mRNA expression in scrambled-shRNA infected MEFs versus shRNA-Tnfaip2-infected MEFs or Infected cells were purified by BFP-sorting on day-4 after infection [n=3 biological replicates; log transformed data were normally distributed (P>0.05 as per Shapiro-Wilk test) and analyzed by t-test with Holm-Sidak for multiple comparisons].RT-qPCR was performed to determine (G) Tnfaip2 mRNA expression in scrambled-shRNA infected MEFs versus shRNA-Vim-infected MEFs. G) Infected cells were purified by BFP-sorting on day-4 after infection [n=3 biological replicates; log transformed data were normally distributed (P>0.05 as per Shapiro-Wilk test) and analyzed by t-test with Holm-Sidak for multiple comparisons].
Figure 11Oct4-eGFP reporter MEFs or WT MEFs were infected with a vector expressing the indicated shRNA in combination with the indicated cDNA (OE indicates overexpression), Cells were co-infected with 4 reprogramming factors (4F= Oct4, Sox2, Klf4, c-myc = OSKM) and double-infected cells were FACS sorted for analysis. (A) RT-qPCR analysis on day-4 after infection to determine RNA expression of Tnfaip2 or Vim in Tnfaip2-depleted cells with ectopic expression of Vim (left) or in Vim-depleted cells with ectopic expression of Tnfaip2 (right). Scramble shRNA (shScr)-infected MEFs served as a control [n=3 biological replicates; log-transformed data were normally distributed (P>0.05 as per Shapiro-Wilk test) and analyzed by t-test].Oct4-eGFP reporter MEFs or WT MEFs were infected with a vector expressing the indicated shRNA in combination with the indicated cDNA (OE indicates overexpression), (B) RT-qPCR analysis on day-4 after infection to determine RNA expression of Vim or Tnfaip2 in Tnfaip2-depleted cells with ectopic expression of Vim (left) or in Vim-depleted cells with ectopic expression of Tnfaip2 (right). Scramble shRNA (shScr)-infected MEFs served as a control [n=3 biological replicates; log-transformed data were normally distributed (P>0.05 as per Shapiro-Wilk test) and analyzed by t-test].Oct4-eGFP reporter MEFs or WT MEFs were (A-D) infected with a vector expressing the indicated shRNA in combination with the indicated cDNA (OE indicates overexpression), ]. (C) Representative images of FACS profiles and (D) quantification of SSEA1+ iPSCs on day 14 after infection of MEF cultures with the indicated combination of shRNAs plus cDNAs (OE) or scrambled shRNA control [n=3 independent cultures per group; data were normally distributed (P>0.05 as per Shapiro-Wilk test) and analyzed by one-way ANOVA and Dunnett's multiple comparison test ]Oct4-eGFP reporter MEFs or WT MEFs were (E,F) infected with the indicated shRNA with or without combined treatment with Etomoxir - a chemical inhibitor of CPT1A. (E) Representative FACS profiles and (F) quantification of the percentage of SSEA1+ iPSCs in MEF cultures on day-18 after infection with shRNAs targeting Tnfaip2 or a scrambled shRNA control with or without continuous treatment with Etomoxir [n=5 biological replicates; data were not normally distributed (P<0.05 as per Shapiro-Wilk test) and analyzed by Mann-Whitney U test with Holm Sidak correction for multiple comparisons].
Figure 12(A) TAG levels in exoc3(RNAi)-treated planarians vs. gfp(RNAi)-treated controls as determined by UPLC-MS/MS, [n=4 experimental replicates on pools of 25 planarians per group per experiment; data were normally distributed(C) RT-qPCR analysis of exoc3 mRNA expression in planarians of the indicated genotypes and treatment schedule(D) Representative images of regenerating planarians that were injected with gfp-RNAi, Smed-β-catenin-1-RNAi or Smed-β-catenin-1-RNAi in combination with PA/PC injection Heads and tails were cut on day-10, images were taken on day-17 after amputation. Number ratios in the photographs indicate the total number of animals exhibiting the represented phenotype.(E) Images of body plan maintenance in non-injured planarians that were injected with gfp-RNAi, exoc3-RNAi or exoc3-RNAi in combination with PA/PC injection Number ratios in the photographs indicate the total number of animals exhibiting the represented phenotype.(F) RT-qPCR analysis of marker genes of early stem cell differentiation (prog-1 and prog-2) and late stem cell differentiation (Smed-odc1) in planarians exposed to the indicated treatment regiments on day-38 of the experiment as indicated in panel-B [n=3 biological replicates; log-transformed data were normally distributed (P>0.05 as per Shapiro-Wilk test) and analyzed by one-way ANOVA and Dunnett's multiple comparison test].
Figure 1(a) Silver-stained gel image of the affinity-purified complexes of PSPC1, SFPQ and NONO. The respective proteins are marked along with AcTEV protease used for the elution of purified proteins.(f) Western blotting of affinity purified complexes of PSPC1, SFPQ and NONO with SWI/SNF antibodies. cBAF, PBAF, ncBAF specific, as well as common subunits, are marked.
Figure 2(a) Silver-stained gel image of the affinity-purified complexes of ARID1A and ARID1B. The bait and a few other SWI/SNF subunits are marked as per their molecular weight.Heat maps showing the enriched proteins (dNSAFavg*1000) normalized to the bait identified in MudPIT analysis focusing on various (c) SWI/SNF subunitsHeat maps showing the enriched proteins (dNSAFavg*1000) normalized to the bait identified in MudPIT analysis focusing on various (e) PSPs.(f) Western blotting of affinity purified complexes of ARID1A and ARID1B with bonafide paraspeckle components. SMARCA4 was used as a positive control for purification.
Figure 3(a) Halo purification was performed from nuclear extracts of scramble and various knockdown HEK293FRT cells expressing Halo-FLAG-tagged ARID1B. Input and eluted samples were resolved on a gel followed by western blotting with various antibodies. Western with SWI/SNF subunits- SMARCA4 and SMARCD1 were used as the control for purification, while beta-actin was used as a loading control.(e, f) (e) RIP qPCR and (f) PCR analysis performed using an antibody against endogenous ARID1B. for monitoring NEAT1_2 levels. RIP with NONO and IgG was used as a positive and negative control respectively. GAPDH levels were also assessed in the assay. Data are expressed as a percent of Input.
Figure 4(c) smFISH analysis was performed to detect NEAT1 signal in the nucleus. The scale bar corresponds to 10 µm. Graph showing the FISH signal quantification across the knockdowns in comparison to scramble control. The number of nuclei (n) used for quantification is mentioned.
Figure 1Western blots showing changing protein levels on the path to senescence in IMR90 and HUVECs. Passage 6 cells represent the proliferating state, and passages 21 and 34 the senescent state for HUVECs and IMR90, respectively.Super-resolution (gSTED) imaging of HMGB1 distribution in proliferating HUVEC nuclei counterstained with DAPI. Bar: 2μm.Representative immunofluorescence images of IMR90 and HUVECs (left) showing reduced HMGB1 levels in senescent nuclei; bean plots quantify this reduction (right; N is the number of cells analyzed per each condition/cell type). Bars: 5 μm. *: P<0.01; Wilcoxon-Mann-Whitney test.
Figure 2Genome browser view of HMGB1 ChIP-seq (normalized to input; mean from two replicates) from proliferating HUVEC (red) or proliferating/senescent IMR90 (grey/green) in the TMEM92 locus.
Figure 3Heatmap showing protein (SASP, from http://www.saspatlas.com/; log fold ratio)and gene expression levels (log2FC) of IMR90 SASP-related genes embedded in high-HMGB1 (red) TADs like those in panel C. **: genes bound by HMGB1 in ChIP-seq data are more than expected by chance; P>0.001, hypergeometric test.
Figure 4Immunofluorescence (top), western blot (middle) and RT-qPCR analyses (bottom; mean fold-change ±S.D. from two biological replicates) confirm HMGB1 knockdown in IMR90.Representative images of IMR90 overexpressing HMGB1-GFP, immunostained for p21 and CTCF, and counterstained with DAPI. IMR90 transfected with empty vectors provide a control. Bar: 5 μm.
Figure 5Genome browser views showing HMGB1 sCLIP data (black) along the ASH1L, CCNL2 and TET2 loci; input tracks (grey) provide background levels. *: significantly-enriched peaks. The consensus motif for HMGB1 binding on RNA is also shown (bottom right).Bar graphs showing mean fold-change of selected mRNAs (over ±S.D. from two biological replicates) from ILF3- (purple), RBMX- (white) or PNN-knockdown experiments (blue) in proliferating IMR90. *: significantly different to siRNA controls; P<0.01, unpaired two-tailed Student's t-test. Dashed line indicates no change in expression.
Figure 1A. RNA-seq of whole blood from symptomatic (n=26; median 8 days PIO) and asymptomatic (n=30; median 3 days post-hospital admission) COVID-19 patients at the acute phase of infection (SARS-CoV-2 PCR-positive) was performed. Only samples with RNA integrity number >6 were sent for sequencing and included in the analysis. Volcano plot indicating DEGs between asymptomatic and symptomatic COVID-19 patients, with thresholds of FDR < 0.05 and |FC| > 2. Numbers of over-expressed and under-expressed genes are indicated.
Figure 2B. Network analysis of significant immune mediators between symptomatic and asymptomatic COVID-19 patients. Interactive relationships between the cytokines or chemokines were determined by STRING (Search Tool for the Retrieval of Interacting Genes/ Proteins) analysis, with a confidence threshold of 0.5.
Figure 3C. SARS-CoV-2 specific CD4+ non T follicular helper (TFH) cells from symptomatic (n=5) and asymptomatic COVID-19 (n=5) patients were characterized with flow cytometry based on the expression of IL-17a, IFN-γ, TNF-⍺, IL-4 and IL-10 upon SARS-CoV-2 peptide stimulation. Representative plot overlay of unstimulated (black) on peptide stimulated (red) was performed on asymptomatic patient. Data are presented as Mean ± SD **P<0.01 (Mann-Whitney U test).
Figure 4A, B. (A) IgG and (B) IgM responses were analysed by screening plasma samples of asymptomatic (n=39, median study day 29) and symptomatic (n=57, median study day 31) COVID-19 patients. Data are presented as Mean ± SD ***P<0.001 (Mann-Whitney U test).
Figure 5A. Flow cytometry acquisition of patient whole blood was performed and number of mature and immature neutrophils were compared between asymptomatic (n=16) and symptomatic (n=52) COVID-19 patients and healthy controls (n=17). Data are presented as Mean ± SD *P<0.05; **P<0.01; ***P<0.001 (Kruskal-Wallis test with Dunn's multiple comparison).B. Mass cytometry was performed on PBMCs obtained from acute asymptomatic (n=19) and acute symptomatic (n=37) COVID-19 patients and healthy donors (n=10). Monocytes were characterized based on their CD14 and CD16 expression. Data are presented as Mean ± SD **P<0.01; ***P<0.001 (Kruskal-Wallis test with Dunn's multiple comparison). C. Classical, intermediate and non-classical monocytes were further defined in acute asymptomatic (n=19) and acute symptomatic (n=37) COVID-19 patients and healthy donors (n=10) with inflammatory marker CD169. Data are presented as Mean ± SD **P<0.01; ***P<0.001 (Kruskal-Wallis test with Dunn's multiple comparison).D. Expression data of genes associated with inflammatory monocytes, activated neutrophils and pro-inflammatory cytokines were compared between acute asymptomatic (n=30) and asymptomatic (n=26) COVID-19 patients and were represented by ratio with respect to expression in asymptomatic patients. Bar graphs show DEGs with FDR adjusted P value < 0.05 and fold change values are represented in the log2 scale.E. Pro-inflammatory associated immune mediator levels in plasma fraction samples from first collection timepoint during hospital admission were quantified with 45-plex microbead-based immunoassay. Immune mediator levels were compared between asymptomatic patients (n=48) and symptomatic patients stratified by COVID-19 severity (mild, n=61; moderate, n=43; severe, n=68). Immune mediator levels for healthy controls (HC) (n=23) are indicated by the black dotted line. Patient samples with concentrations out of measurement range are presented as the value of Limit of Quantification (LOQ). Data are presented as Mean ± SD *P<0.05; **P<0.01; ***P<0.001 (One-way ANOVA with post hoc t test).
Figure 6A. Growth factors in the plasma of asymptomatic (n=48), symptomatic patients (n=173) and healthy controls (n=23) were analyzed by Luminex. A ratio of VEGF-A/VEGF-D was calculated and used to perform receiver operating characteristics analysis. The area under the curve was shown. Data are presented as Mean ± SD **P<0.01; ***P<0.001 (Kruskal-Wallis test with Dunn's multiple comparison).B. RNAseq analysis was performed on whole blood and selected transcripts that are overexpressed in asymptomatic patients over symptomatic patients are shown. Genes with false discovery rate less than 0.05 (*), 0.01 (**) or 0.001 (***) are indicated.C. Cell counts of DCs, CD4 T cells, CD8 T cells and B cells that express specific chemokine markers between asymptomatic (n=19), symptomatic patients (n=37) and healthy controls (n=10) are analyzed by CyTOF and shown. Data are presented as Mean ± SD. *P<0.05; **P<0.01; ***P<0.001 (Kruskal-Wallis test with Dunn's multiple comparison).