EMBO/SourceData huggingface dataset:
Figure 6a, b) AAV-hACE2 mice were treated with saline and challenged with SARS-CoV-2 (8 × 104 PFU) for 5 days. Samples were collected at day 5 post-infection, and the collagen were stained with Picro Sirius red (a), while collagen deposition was quantified using MetaMorph software and presented as area (μm2) (b). The images were captured by light microscopy (upper panel) and polarized light microscopy (lower panel). Data are mean ± SEM from two independent experiments (saline: n=3 for 3 d.p.i., n=4 for 5 d.p.i.; isotype: n=3 for 3 d.p.i., n=4 for 5 d.p.i.; prophylactic treatment of CLEC2.Fc treatment: n=5 for 3 d.p.i. and 5 d.p.i.; therapeutic treatment of CLEC2.Fc: n=3 for 3 d.p.i. and 5 d.p.i.). ***p < 0.001, ****p < 0.0001 (Two-way ANOVA).
Figure 1(C-F) Two months after a single tail vein injection of rAAV, wild-type mice showed similar body weight (C), blood glucose levels (D), serum alanine aminotransferase (ALT) (E) and aspartate aminotransferase (AST) activities (F) with the mice injected with PBS. n = 8 - 10 for each group.(H-I) Two months after a single injection of rAAV, db/db mice showed similar body weight (H) and blood glucose levels (I) with the mice injected with PBS. n = 10 for each group. (J-K) Serum ALT (J) and AST (K) of mice in (H).(M) The hepatic p-MLKL protein level in normal and necrotic area from mice injected with PBS or rAAV in (H). Ncp, Necroptosis. n = 7 - 8 biological replicates.(N) The hepatic mRNA levels of Il-6, Il-1β, Tnf-α and Mcp-1 involved in necroptosis at normal or necroptotic area from mice injected with PBS or rAAV in (H). n = 7 - 8 biological replicates.(Q-R) Six months after a single injection of rAAV, db/db mice showed decreased body weight (Q) and similar blood glucose levels (R) compared to the mice injected with PBS. n = 8 - 12. (S-T) Serum ALT (S) and AST (T) activities of mice in (Q). n = 8 - 10.
Figure 2Body weight (B) were measured 6 weeks later, after injection once a day for 5 consecutive days with vehicle (Veh) or streptozotocin (STZ). n = 6 - 19 for each group.blood glucose levels (C) were measured 6 weeks later, after injection once a day for 5 consecutive days with vehicle (Veh) or streptozotocin (STZ). n = 6 - 19 for each group.Two months after a single injection of rAAV, hyperglycemic mice induced by streptozotocin showed , blood glucose levels (F) compared with those mice injected with PBS. n = 9 (G-H) Serum ALT (G) and AST (H) activities of mice in (D).(J) The hepatic p-MLKL level of mice in (D).Body weight (L) of RHH (Resistance to HFD-induced Hyperglycemia) mice fed with HFD for 15 weeks. n = 6 - 12.blood glucose levels (Q) of obese and euglycemic mice in (L) after a single injection of PBS or rAAV for 2 months. n = 6. Serum ALT (R) of mice in (O).(T) Liver images, H&E staining of liver sections and incidence of hepatic necroptosis for mice in (O).(U) The hepatic p-MLKL level of mice in (O).
Figure 3Two months after a single injection of PBS or rAAV blood glucose levels (G) of hyperglycemic and obese mice in (B) were not significantly changed. n = 10 - 11 for each group. (H-I) Serum ALT (H) and AST (I) activities of mice in (E).(K) The hepatic p-MLKL protein level in normal or necrotic area from mice injected with PBS or rAAV in (E).(L) The hepatic mRNA levels of Il-6, Il-1β, Tnf-α and Mcp-1 involved in necroptosis at normal or necroptotic area from mice in (E).blood glucose levels (O) of hyperglycemic and obese mice after a single injection of PBS or rAAV for 6 months. n = 8. (P-Q) Serum ALT (P) and AST (Q) activities of mice in (M).(R) Liver images, H&E staining and ki67 immunostaining of liver sections and HCC incidence of mice in (M).
Figure 4(A) Prednisone treatment markedly blocked the increase of some key inflammatory factors in mouse macrophages treated with poly(I:C) (pIC). n = 3 in duplicate.Prednisone treatment markedly blocked the increase of p-MLKL level (C, n = 3 in duplicate.) of macrophages induced by poly(I:C) and z-VAD-fmk (zVAD). Scar bar, 100 μm.(J) Representative liver images for mice in (E).(M) The hepatic p-MLKL level of mice in (E).
Figure 5(A) The top 15 hepatic genes upregulated in 9-week-old db/db mice compared with wild type mice. n = 1, liver samples from 3 mice were mixed together in equal amounts for RNA extraction.Body weight (D) of db/db mice after a single injection of rAAV-si-NC/Cyp4a14/Pebp1/Tat for 2 months. n = 10 - 12. rAAV-si-NC was packaged as indicated in Fig. 1A(I) Liver images, H&E staining of liver sections and the incidence of death and hepatic necroptosis (Ncp) of mice in (D). n = 10 - 14, NML, Normal.(K) The hepatic p-MLKL level of the indicated mice in (D).(L) The indicated hepatic mRNA levels of mice in (K).
Figure 6Two months after a single injection of rAAV-si-Pebp1, HFD-induced hyperglycemic and obese mice showed blood glucose levels (C) with the mice injected with rAAV-si-NC. n = 10 - 11. (D-E) Serum ALT (D) and AST (E) activities from mice in (B). The colored dots indicate the mice with high ALT or AST activity, and the same color dot in (D) and (E) indicates the same mouse.(G) The hepatic p-MLKL level of mice in (B).(H) The indicated hepatic mRNA levels of mice in (B).
Figure 7(A) The effect of si-Pebp1 in primary mouse macrophages. n = 3. (B) Knockdown of Pebp1 markedly blocked the increase of some key inflammatory factors in mouse macrophages treated with poly(I:C). n = 6.Knockdown of Pebp1 markedly blocked the loss of cell membrane integrity (C, n = 6 in duplicate or triplicate.) of mouse macrophages induced by poly(I:C) and z-VAD-fmk.(E) Knockdown of Pebp1 attenuated the increase of p-Tbk1 level induced by poly(I:C). n = 3 biological replicates.Knockdown of Tbk1 markedly blocked the loss of cell membrane integrity (H of mouse macrophages induced by poly(I:C) and z-VAD-fmk.After a single injection of rAAV-si-Tbk1 for 11 weeks, the db/db mice showed blood glucose levels (M) with the mice injected with rAAV-si-NC. n = 11. (N-O) Serum ALT (N) and AST (O) activities of mice in (K).(R) The hepatic p-MLKL level of mice in (K).
Figure 1B, Immunofluorescence staining of single nuclei for H3K79me3 or H3K79me2 (green) and the pericentromeric heterochromatin marker HMGA1 (red). Upper panels, prometaphase; lower panels, interphase. DNA is stained with DAPI (blue). Scale bar, 2µm.H, H3K79me2 and H3K79me3 ChIP-seq enrichment relative to input at selected repetitive element sequences.K, Immunofluorescence staining for H3K79me3 in control and Dot1l KO nuclei. Scale bar, 2µm.M, DNA Fluorescence in situ hybridization (DNA FISH) for the mouse major satellite element with immunofluorescence staining for H3K79me3 (top) or H3K79me2 (bottom) in single mESC nuclei. Scale bar, 5µm. Plot shows Pearson's correlation values between FISH and H3K79me2 or H3K79me3 signal, each point representing an individual nucleus. ***p<0.0001, two-tailed unpaired t-test.
Figure 2B, Metagene plots showing mean ChIP signal for H3K79me2 and H3K79me3 at downregulated and upregulated genes identified in (A).F, RNA FISH for major satellite transcripts and simultaneous staining for H3K79me3 in control and Dot1l KO mESCs. Scale bar, 5µm. Below left, quantitation of fluorescence intensity along a line bisecting a representative nucleus. Bisecting lines used to generate the plots are shown in the merged images. Below right, quantitation of total FISH fluorescence signal for n=46 cells of each genotype. Line summaries show mean ± standard deviation. ***p<0.0001, unpaired Welch's t-test.
Figure 3A, ChIP-qPCR for HP1β at selected repeat elements in Dot1l KO and wild type mESCs. Bars represent mean of n=2 biological replicates. Neg. control indicates an intergenic locus (mm10 chr19:51758385-58758494) not strongly associated with either euchromatin or heterochromatin.B, Immunofluorescence staining of HP1β (green) in wild type and Dot1l KO mESCs. DNA is stained with DAPI (blue). Quantification of signal is shown at right. Each point represents one nucleus (Dot1l KO n=83 and control n=104 nuclei). Scale bar, 10µm. ***p<0.0001, Mann-Whitney U test.G, Western blot for co-immunoprecipitation of DOT1L and SMARCA5 in mESCs. AF10 is a positive control and GAPDH is a loading control.H, Immunofluorescence staining of SMARCA5 (green) and HMGA1 (red) in mESCs, indicating localization of SMARCA5 to heterochromatic regions. Arrowheads indicate an example of SMARCA5 enrichment at PCH. DNA is stained with DAPI (blue). Scale bar, 4µm. Bottom, quantitation of fluorescence intensity in each channel along a line bisecting the nucleus.J, H3K79me3 enrichment at major satellite sequences in Smarca5 knockdown (KD) relative to scrambled shRNA control mESCs measured by ChIP-qPCR. Bars represent mean of n=3 biological replicates and error bars represent ± SD. p=0.0876 for the major satellite element (one-sample t-test compared to expected fold change of 1, shown as a dashed line).L, Immunofluorescence staining of H3K79me3 (red) in mouse 3T3 fibroblasts transduced with a vector expressing GFP and a short hairpin against Smarca5 (shSmarca5) compared to untransduced control. Arrowheads indicate DAPI-dense chromocenters. Merge includes GFP channel for transduced cells. Scale bar, 10µm.
Figure 4A, Immunofluorescence staining of H3K79me3 (green) and H3S10P (red) in a single nucleus of control and Dot1l KO mESCs. DNA is stained with DAPI (blue). Scale bar, 5 µm.B, Example metaphase spreads in wild type and Dot1l KO mESCs showing examples of a chromosome break, premature centromere separation, and chromosome fusion (yellow boxes). Scale bar, 5 µm.F, Western blot for the DNA damage marker γH2A.X in control and Dot1l KO mESCs. Quantitation of signal on the blot for n=2 biological replicates is shown.
Figure 5A, Immunofluorescence staining of H3K79me3 (green) and H3K9me3 (red) in wild type mouse embryos during the first 48 hours of preimplantation development. Bottom images show an embryo treated with DOT1L inhibitor at 48 hours post fertilization (hpf). DNA is stained with DAPI (blue). Scale bars, 10 µm (main image) or 4 µm (inset). Quantitation of pixel-by-pixel fluorescence intensity for H3K9me3 and H3K79me3 for a representative embryo is shown at right. AU, arbitrary units.B, Quantitation of numbers of total, H3K9me3-positive, and H3K79me3-positive chromocenters in control and inhibitor-treated embryos at 48 hpf. Chromocenters were defined as DAPI-dense foci. Each point represents one nucleus. N=69 (control) or n=20 nuclei (inhibitor treated) in n=19 (control) or n=5 (inhibitor treated) embryos *p<0.05, ***p<0.001, two-tailed unpaired t-test.
Figure 6A, Volcano plot showing differentially expressed single-copy genes in DOT1L inhibitor-treated vs control embryos at 56hpf (8 cell stage). Each dot signifies one gene. Differentially expressed genes (adjusted p-value ≤ 0.05; no threshold for log2 fold change) are shown in red. P-values were corrected for multiple hypothesis testing using the Benjamini-Hochberg method.
Figure 1Percentage of 2-cell-like cells obtained after hyperosmotic treatments for 24h (B) Shown are the mean ± s.d. from at least 2 replicates. Individual dots indicate measurement of an independent biological replicate by FACS. Unpaired two-tailed t-tests were performed. Data information: Stars indicate significant differences obtained using indicated statistical tests with *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.Percentage of 2-cell-like cells obtained after hyperosmotic treatments the indicated time (C-D). Shown are the mean ± s.d. from at least 2 replicates. Individual dots indicate measurement of an independent biological replicate by FACS. Unpaired two-tailed t-tests were performed. Data information: Stars indicate significant differences obtained using indicated statistical tests with *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.H-I. Protein levels assessed by Western blotting in the indicated conditions. Shown is one representative image from 2 independent replicates.
Figure 2A. (Left) Representative single confocal z-section of CellROX-DeepRed fluorescence of ESC treated with 0.2M sorbitol for the indicated time from 2 biological replicates. Scale bar: 10µm. (Right) Quantification of CellROX-DeepRed fluorescence intensity from single z-sections from 2 biological replicates. Two-tailed Mann-Whitney tests were performed. Boxes indicate the range between the first and third quartile, the line indicates the median and the whiskers display the spread from 5 to 95% of the data. Data information: Stars indicate significant differences obtained using indicated statistical tests with *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.B. Percentage of 2CLCs obtained by FACS after the indicated sorbitol treatment with addition of ROS scavenger N-AcetylCysteine (NAC) during either the sorbitol treatment or recovery time. Shown are the mean ± s.d. from at least 2 replicates. Individual dots indicate measurement of an independent biological replicate by FACS. Unpaired two-tailed t-tests were performed. Data information: Stars indicate significant differences obtained using indicated statistical tests with *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.C. Protein levels assessed by Western blotting in the indicated conditions. Shown is one representative image from 3 independent replicates.D. Percentage of 2CLCs obtained by FACS after the indicated hyperosmotic treatment with addition of ATR inhibitor during either the hyperosmotic stress or recovery time. Shown are the mean ± s.d. from at least 2 replicates. Individual dots indicate measurement of an independent biological replicate by FACS. Same untreated dots are shown. Two-tail t-tests were performed. Data information: Stars indicate significant differences obtained using indicated statistical tests with *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.
Figure 3A. Percentage of 2CLCs obtained by FACS after the indicated sorbitol treatment with addition of ROS scavenger N-AcetylCysteine (NAC) or ATR inhibitor. Shown are the mean ± s.d from 5 replicates. Individual dots indicate measurement of an independent biological replicate by FACS. Unpaired two-tailed t-tests were performed. B. Percentage of 2CLCs obtained by FACS after the indicated NaCl treatment with the addition of ROS scavenger N-AcetylCysteine (NAC) or ATR inhibitor. Shown are the mean ± s.d. from at least 2 replicates. Individual dots indicate measurement of an independent biological replicate by FACS. Unpaired two-tailed t-tests were performed. Data information: Stars indicate significant differences obtained using indicated statistical tests with *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.C. Percentage of 2CLCs obtained by FACS after the indicated treatments. Shown are the mean ± s.d from at least 2 replicates. Individual dots indicate measurement of an independent biological replicate by FACS. Individual and combinatorial treatments performed with 0.2M of sorbitol, 24mM of sodium acetate, 2.5nM of PlaB spliceosome inhibitor and 0.53µM of retinoic acid. Unpaired two-tailed t-tests were performed. Unpaired two-tailed t-tests were performed against each individual treatment in the combinatorial experiments. Data information: Stars indicate significant differences obtained using indicated statistical tests with *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.
Figure 2L) Example images of β-Gal+ traced cells post-irradiation and at homeostasis. Scale bar= 100um. M) The number of fully traced β-Gal+ crypt-villus axes/cm in irradiated and non-irradiated mice. Lineage tracing events were quantified from small intestines divided into four equal segments (S1-4, with S1 being the most proximal), stained with X-Gal, embedded in paraffin, cut into 5um thick sections, and counterstained with neutral red. Data are expressed as mean ± SEM. N=3 mice/group, P-value generated by multiple student's t-tests. N) The number of β-Gal+ fully traced crypt-villus axes (i.e. lineage tracing events) normalized to the number of pre-injury crypt marked β-Gal+ cells (quantified from d0 control mice). S1 + S2 = the two most proximal intestinal segments. Data are expressed as mean ± SEM. N=4 mice/group, P-value generated by two-tailed student's t-test.
Figure 3D) Organoid formation efficiency of different aged LRC populations. N=3 mice/group, N=3 wells quantified per mouse. Data are expressed as mean ± SEM. N=3 mice/group,E) Images of organoids quantified in (D).
Figure 4C) Western blot demonstrating LC3 levels from equal numbers of sorted CytoIDlow and CytoIDhigh cells. 200,000 cells were loaded per lane.E) Representative images of organoids formed from sorted CytoIDlow, CytoIDhigh, and Lgr5-GFPhigh cells. F) Quantification of the organoid-formation efficiency in (E). N=4 mice/group, N=3 wells quantified per mouse. P-Values displayed on graph. Data are expressed as mean ± SEM. P value generated by ratio paired two-tailed t-test.G) Effect of bafilomycin on organoid formation efficiency in CytoIDlow and CytoIDhigh cells. Cells were plated in media containing different concentrations of bafilomycin (0, 2.5, 5, 10, or 25 ng/mL) on day 0, media was changed on day 3 to media that did not contain bafilomycin and organoids were imaged on day 5 after plating. N=4 mice/group, N=4 wells quantified per mouse.H) Representative images of organoids quantified in (G).
Figure 6A) Representative plots demonstrating the distribution of cells with and without CytoID puncta within sorted CytoID low, high, Chga+, and c-Kit+ cells.B) Quantification of the percentage of cells with and without puncta within CytoID low, high, Chga+, and c-Kit+ cells. N=3 mice/group (biological replicates).C) Representative images and quantification of organoid-formation efficiency of CytoID low and high cKit+ and Chga+ cells. Scale bars = 500um. N=3 mice/group (biological replicates), N=4 wells quantified per mouse (technical replicates).D) Immunofluorescent images of crypts from CAG-LC3-GFP-RFP and Lgr5-GFP mice 6h post-irradiation. Sections were stained for γH2AX, E-cadherin, and either LC3-RFP or Lgr5-GFP respectively. E) Quantification of the number of γH2AX foci in cells with RFP+ puncta, without autophagic puncta, or in Lgr5+ CBCs. N=3 mice/group, 40-80 cells quantified/mouse.
Figure 1C Immunostaining for the naive pluripotency marker KLF17 and the shared pluripotency marker OCT4. Scale bar: 50μm. Data information: Representative images of n=3 biological replicates are shown.D Gene-expression analysis by qPCR of KiPS primed-iPSCs, KiPS-chemical resetting at day 14, and KiPS naive-iPSCs. Data information: Bars indicate the mean ± SEM of n=3 biological replicates shown as dots. (D) Expression was normalised to the mean of HPD06 naive-iPSCs samples.I Barplots showing the absolute expression (CPM) of twelve trophoblast genes found to be significantly UP-regulated in KiPS-chemical resetting vs KiPS primed-iPSCs comparison. Data information: Bars indicate the mean ± SEM of n=3 biological replicates shown as dots. ***P < 0.001, ****P < 0.0001, ns no significance (Student's t-test).
Figure 2B Gene expression analysis by RT-qPCR for naive (KLF17, TFCP2L1, and NANOG) and trophoblast (GATA3, GATA2, KRT7, ELF5, TP63, and TFAP2A) marker genes in naive-iPSCs and TSC cells derived from two different naive-iPSC lines (HPD06 and H9).I Gene-expression analysis by qPCR of KiPS primed-iPSCs, KiPS in PXGL medium at day 14, KiPS in TSC medium at day 14, H9 naive-TSCs on ColIV, H9 naive-TSCs on MEF and HPD06 naive-iPSCs. Data information: (I) Dots indicate biological replicates and bars indicate the mean ± SEM of n=3 biological replicates. Top: naive, primed, and general pluripotency markers, expression was normalised to HPD06 naive iPSCs; bottom: trophoblast markers, expression was normalised to H9 naive-TSCs.J Immunostaining for the pluripotency markers OCT4 and KLF17, and the TSC markers GATA3 and KRT7 of HPD06 naive-iPSC and ccTSC.01 cultured on MEF and on collagen IV (ColIV) after 3 passages. Scale bars: 30 μm. Data information: Representative images of n=2 biological replicates are shown.
Figure 3B Immunostaining for the pluripotency markers OCT4, and the TSC markers GATA3 and KRT7 of KiPS primed-iPSCs, HPD06 naive-iPSCs, ccTSC.02 at passage 4, ccTSC.01 at passage 4 and HPD06 naive-TSCs. Scale bars: 30 μm. Data information: (B) Representative images of n=3 biological replicates are shown.F Barplots showing the absolute expression as log(CPM+1) of ADAP2, HAVCR1, SLC28A3, ARID5B, and GATA2 in the reported conditions. Data information: (F) Bars indicate the mean ± SEM of at least n=5 biological replicates from at least 2 independent studies shown with different symbols. ***P < 0.001, ns no significance (Student's t-test).
Figure 4A Gene-expression analysis by qPCR of syncytiotrophoblast (left) and extravillous (right) markers. Data information: (A) Dots indicate biological replicates. Left: expression was normalised to HPD06 naive-STBs and ccTSC.02-STBs; Right: expression was normalised to HPD06 naive-EVTs and ccTSC.02-EVTs.B Top: Immunostaining for the TSC marker KRT7 and the STB marker SDC1 of ccTSC.02-STBs, HPD06 naive-STBs, ccTSC.02, and HPD06 naive-TSCs. Scale bars: 50 μm. Bottom: Immunostaining for the TSC marker KRT7 and the EVT marker HLA-G of ccTSC.02-EVTs, HPD06 naive-EVTs, ccTSC.02, and HPD06 naive-TSCs. Scale bars: 25 μm. Data information: (B) Representative images of n=2 biological replicates are shown.
Figure 5D Barplots showing the absolute expression as log(CPM+1) of general pluripotency (OCT4 and NANOG), naive (SUSD2), and TSC-specific genes (GATA2, GATA3, KRT7, and ADAP2) in the reported conditions highlighted in different colours. Data information: (D) Dots indicate biological replicates and bars indicate the mean ± SEM of n=3 biological replicates.
Figure 6B Immunostaining for the TSC markers GATA3 and KRT7, the naive pluripotency marker SUSD2 and general pluripotency markers OCT4 of KiPS primed-iPSCs, KiPS at day 3 of the conversion, KiPS at day 7 in TSC medium or PXGL, KiPS at day 14 in TSC medium or PXGL, HPD06 naive-TSCs and HPD06 naive-iPSCs. Scale bars: 25 μm. Data information: (B) Representative images of n=2 biological replicates are shown.C Single-cell scatter plots showing the log(CPM+1) integrated intensity of TSC markers GATA3 and KRT7, naive pluripotency marker SUSD2, and general pluripotency marker OCT4 of KiPS primed-iPSCs, KiPS at day 3 of the conversion, KiPS at day 7 in TSC medium or PXGL, KiPS at day 14 in TSCs medium or PXGL, HPD06 naive-TSCs and HPD06 naive-iPSCs. Black lines represent the threshold used to define positivity to a given marker. Data information: (C) Fluorescence intensity was measured by immunofluorescence in 5 to 12 independent fields from 2 independent experiments.
Figure 1D-F. Volcano plots showing RNA-seq data in mouse islets (20% vs 5% O2 for 24 hours; n = 3 mice/group; D), human islets (20% [n = 2 biological replicates from donor 1 sample] vs 2% [n = 3 biological replicates from donor 1 sample] O2 for 24 hours; E), and MIN6 cells (20% vs 5% O2 for 6 hours; n = 3 biological replicates; F). Atf3, Bhlhe40, and other reported hypoxia-inducible transcriptional repressor genes (Cavadas et al., 2017) are shown (red, significantly upregulated genes).