EMBO/SourceData huggingface dataset:
Figure 7a. mRNA expression on ex vivo isolated Tregs from MS patients and healthy controls (n=15).b. Representative example of the expression of AKT3, Foxo3 and PTEN on ex vivo sorted Tregs from MS patients (black open histogram) and HC (grey open histogram). Grey histograms represent isotype. c. Ex vivo expression of pAKT, pFoxo1 and pFoxo3 in Tregs from RRMS patients (black open histogram) and healthy controls (white open histograms). Grey histograms represent isotype.d. Representative example of the frequency of IFN+Foxp3+ Tregs isolated ex vivo from RRMS patients (lower row) and healthy controls (HC, upper row) and stimulated in the presence of vehicle (left histogram), LY294002 (middle histogram) or MK2206 (right histogram) for 3 days. Staining was performed after a 4-hour stimulation with PMA and ionomycin. e. Statistical analysis of the frequency of IFN+Foxp3+ Tregs in HC (white bars) and RRMS patients (black bars) stimulated in the presence of either vehicle, LY294002 or MK2206 (n=10).f. Representative example of the proliferation of Tresp cells from HC (upper row) and RRMS patients (lower row) either alone (first column) or in co-culture with Tregs that had been previously incubated with either vehicle, LY294002 or MK2206 for one hour and washed to remove drug traces. g. Statistical analysis of the percentage of suppression as in h at different Treg:Tresp ratios of n=8 experiments performed. *p < 0.05, **p < 0.005, ***p < 0.0005.
Figure 1B Representative examples of chromatin accessibility and gene expression of KRT5 and KRT10, two key epidermal differentiation-related genes, in the main keratinocyte populations.C Co-accessibility at the human epidermal differentiation complex (EDC) in undifferentiated (cluster 2) and differentiated (cluster 3) keratinocytes. Only connections (arcs) with a co-accessibility score above 0.25 are plotted. Grey boxes below tracks represent scATAC-seq peaks.D Left: Heatmap displaying the differentially accessible peaks between undifferentiated (cluster 2) and differentiated (cluster 3) keratinocytes. Number of Tn5 insertion sites in each region were scaled by row. Right: DNA sequence motifs for the top six overrepresented transcription factor (TF) motifs in undifferentiated (cluster 2) and differentiated (cluster 3) keratinocyte-specific accessible peaks.E Heatmaps displaying the predicted top five transcription factors in each cell cluster using chromVAR motif activity (left) and gene expression (right). F chromVAR deviations (in quantiles) and gene expression for representative enriched TF projected onto the joint UMAP plot of keratinocytes from the single-cell multi-omics dataset.G Heatmap showing the top five active regulons in each cell cluster, based on the expression data of the multi-omics dataset. TF regulons also identified by other approaches are highlighted.
Figure 2A Average histone modification profiles of undifferentiated and differentiated keratinocyte-specific peaks using previously published data generated on NHEK cells (ENCODE). The normalized signal of H3K27ac, H3K4me1/me2/me3 and H3K27me3 were measured in a window of ±10,000 base-pair (bp).B Fractions of CpGs located within epigenomic substructures for the 4,351 InfiniumEPIC CpG probes found within undifferentiated and differentiated keratinocyte-specific peaks.C Unsupervised hierarchical clustering of 12 healthy, 20 AK and 35 cSCC epidermal samples based on the methylation status at undifferentiated and differentiated keratinocyte-specific peaks. Each row represents the average methylation value of all CpGs contained in a particular peak.
Figure 3B, C Average DNA methylation in the 15 chromatin states defined by ChromHMM in NHEK for (B) both EpSC and Keratinocytes from the sci-MET single-cell dataset, or for (C) EpSC-like and keratinocyte-like AK/cSCC from the EPIC dataset. ChromHMM state 1: active promoter; state 2: weak promoter; state 3: inactive/poised promoter; state 4: strong enhancer; state 5: strong enhancer; state 6: weak/poised enhancer; state 7: weak/poised enhancer; state 8: insulator; state 9: transcriptional transition; state 10: transcriptional elongation; state 11: weak transcription; state 12: polycomb-repressed; state 13: heterochromatin/low signal; state 14: repetitive/copy number variation; state 15: repetitive/copy number variation. AK: actinic keratosis, cSCC: cutaneous squamous cell carcinoma. Data information: Statistical analyses in B and C were performed using a Wilcoxon Rank Sum test comparing the average methylation values in genomic states 4 and 5 between EpSC (n= 6) and keratinocytes (n= 548) (B) or EpSC-like (n= 22) and keratinocyte-like (n= 33) AK/cSCC samples (C). *P <0.05, **P <0.01, ***P <0.001, ****P<0.0001.D Heatmaps displaying the gene expression (upper) and the imputed promoter DNA methylation (lower) reference matrices for keratinocyte populations involved in terminal differentiation, calculated by EpiSCORE. The weight of each gene in the reference DNA matrix is also depicted.
Figure 4C Upper: Heatmaps displaying the unsupervised clustering of 67 AK/cSCC and healthy controls based on the methylation patterns of the MIR200C/141 cluster and the MIR205 gene. Lower: Bar plots showing the quantification of the methylation values at CpGs located at promoter regions in each cell-of-origin-based subclass. For the MIR200C/141 cluster, probes located at the regulatory CpG island are depicted with green arrows. For the MIR205 gene, all probes shown in the heatmap are part of the promoter region and were used for quantification. Data information: AK: actinic keratosis, cSCC: cutaneous squamous cell carcinoma.D Left: Representative images of EpSC-like (n=5) and Keratinocyte-like (n=5) cSCC tumors stained for the EMT-marker ZEB2 (green) and the keratinocyte marker TP63 (red). Nuclei were counterstained with DAPI. Images are shown at 40x original magnification. Scale bar, 50 µm. Right: Quantification of ZEB2-positive tumor cells (TP63-positive) in three independent regions per sample. Each dot represents a region with at least 500 tumor cells (TP63-positive) counted. Data information: Statistical analysis in D was performed using an unpaired two-sided t-test (EpSC-like: n= 5 samples, 15 technical replicates; keratinocyte-like: n= 5 samples, 15 technical replicates). Central bar represents the mean and error bars represent the standard deviation. *P <0.05, **P <0.01, ***P <0.001, ****P<0.0001.
Figure 1 (D, E) Representative examples of dose-response curves and time courses. Lines through the dose-response curves are fits to the Michaelis-Menten equation; time courses are fitted to linear equations. Shown are means and standard errors over at least three independent repeats. (F) Fold increase (positive values) or decrease (negative values) in the initial uptake rates of the variants relative to WT GltPh. Circles are averages of technical triplicates. Bars are means over independent repeats (as indicated). The dashed line marks a four-fold increase in the initial rate.
Figure 2 (A, B) Schematic representations of the experimental conditions are shown on the far left. Data are shown for WT GltPh and select gain-of-function mutants, as indicated above the panels. Transition density plots (top of each panel) show the frequency of transitions between the EFRET values. The number of trajectories analyzed (N) and the population-wide mean frequency of transitions is shown on the panels. Representative 50 s sections of single-molecule EFRET trajectories (middle) with raw data in black and idealizations in brown. Scale bar and the conformational states corresponding to the low and intermediate EFRET values are shown to the left of the panels. Dwell-time distributions for the OFS in blue and the IFS in red (bottom). Mean dwell times are shown on the panels in corresponding colors. Black lines represent photobleaching survival plots normalized from 1 to 0. Shown are means and standard errors over at least three independent repeats.
Figure 3(A) Changes in the initial rates of substrate uptake (gray bars), transport domain dynamics (black bars), and l-Asp dissociation constant (white bars) of the mutant GltPh variants relative to the WT transporter. The transition frequencies were measured under non-equilibrium transport conditions, and frequencies obtained in the presence of d,l-TBOA were subtracted from the data. Error bars represent standard errors for at least three independent measurements.
Figure 4(A) Dwell-time distributions of the OFS (blue) and the IFS (red) for WT (top) and K290A (bottom) GltPh observed under equilibrium conditions in saturating Na+/l-Asp (left). Lines are fits to three exponentials. Data are averages and standard errors of at least three independent measurements. (B) Representative EFRET trajectories of WT GltPh showing different transition frequencies. Raw data are in black, and idealizations are in brown. Scalebar is above the trajectories.  (C) Distribution of the WT (top) and the K290A GltPh (bottom) molecules with different mean transition frequencies (white bars). N is the number of trajectories longer than 90 s used in the analysis. The stacked black bars are fractions of trajectories without transitions. Pink bars show the expected binomial distribution if all trajectories shared the mean transition frequency of 0.04 s-1 for the WT and 0.34 s-1 for the K290A. Data are averages and standard errors of at least three independent measurements. (D) 2D-histograms of the consecutive dwell lengths in the OFS (top) and the IFS (bottom). From left to right: calculated distribution of dwell times randomly selected from the distributions in panel A; measured distribution for WT GltPh and the K290A mutant.(E) Representative trajectories of the WT (left) and the K290A (right) GltPh molecules showing switching between dynamic modes. Raw data are in black and idealizations are in brown (top). Black and grey bars under the trajectories indicate apparent slower and faster dynamic modes. Survival plots for the OFS and the IFS (middle). Solid lines are fits to a single (IFS) and double (OFS) exponentials. Dashed lines (OFS only) are rejected fits to single exponentials. Autocorrelation plots of the sequential dwell durations (bottom).
Figure 5(A, B) 2D-histograms of lifetime pairs obtained for trajectories exhibiting single exponential behavior. N is the number of traces used. Scale bar shows relative density normalized by the number of molecules. Above and to the right of each panel are stacked histograms of, respectively, the OFS and the IFS lifetimes of the analyzed molecules (open bars) and the photobleaching time of molecules showing no transitions (black bars). Data from three independent measurements were combined for presentation. (C) The mean lifetimes of the molecules falling within 30 % of the most populated bins of the 2D-histograms (yellow to red). The open blue bar represents the calculated mean OFS lifetime for WT GltPh Shown are means and standard errors of the mean of at least three independent measurements.
Figure 1A, B Temporal expression of Snail genes in the intestinal tract in (A) embryonic E12.5 to E18.5 and (B) postnatal P0 to P20 measured by qRT-PCR. Shown are means ± SD.C, E, G, I Immunohistochemical staining for Snai1/2 (Abcam ab85931) (C) E14.5midgut, (E) E14.5hind gut, (G) postnatal day 14 SI, (I) postnatal day 14 colon. Snai1immunohistochemistry peptide competition controls are shown as inserts (C', E', G', I'). Scale bars: 20 mm.D, F, H, J, K X‐gal staining for β‐galactosidase activity in tissue from Snai2LacZ mice stained in whole mount, sectioned and counterstained with nuclear fast red (D) E14.5midgut, (F) E14.5hind gut, (H) postnatal day 14 SI, (J) postnatal day 14 colon, (K) adult SI. Controls for X‐gal staining are shown as inserts (D', F', H', J').L, M, N Double immunostaining of small intestinalcrypts for Snai1/2 (red) and (M) Lgr5‐GFP (green); (N) merge shows Snai1 is present in both CBCstem cells and proliferating transit amplifying cells. Scale bars: 10 mm.O, P Immunohistochemistry for Snai1 using a different primary antibody (Genetex GTX125918) shows minimal expression of Snai1 in villi. (P) Expression of Snai1 is detected at the base of crypts (P) with strong nuclear expression in CBCstem cells indicated by arrows. Scale bars: 20 mm (O), 10 mm (P).Q Schematic diagram showing Snai1 expression (red) within different cell types in the crypt. SI = small intestine. See also Supplementary Fig S1.
Figure 2Periodic Acid Schiff (PAS) staining was used to detect goblet cells in sections of small intestine from control and Snai1 KO mice. Graph shows number of goblet cells per crypt-villus axes (n = 3).Immunohistochemistry for synaptophysin was used to detect enteroendocrine cells (marked by arrows). Quantification of enteroendocrine cells per crypt-villus axes in control and Snai1 KO tissue revealed a significant increase in the number of enteroendocrine cells following Snai1 loss (n = 3, P = 0.004).Immunohistochemistry for lysozyme was used to detect Paneth cells. Quantification of Paneth cells in control and Snai1 KO indicates a significant increase in Paneth cell numbers following knockout of Snai1 (n = 4, P = 0.002).Measurement of villus length (μm) (n = 3, P = 0.013) and quantification of enterocytes per crypt-villus unit demonstrate a significant reduction in absorptive enterocytes following Snai1 loss (n = 3, P = 0.036).Measurement of villus length (μm) (n = 3, P = 0.003) 8 days after β‐NF induction demonstrates a significant shortening of villi. Bars represent mean ± SD. Two‐tailed Student's t‐test was used to assess significance. Scale bars: 20 μm.
Figure 3Sections from control and Snai1 KO small intestine were stained for PCNA to detect proliferating cells (arrows depicting proliferating CBC stem cells at the base of crypts). Quantification of total PCNA‐positive cells per crypt and PCNA‐positive cells in the base of crypts both revealed a significant reduction in the number of proliferating cells after conditional depletion of Snai1 (n = 4, P = 0.049, 0.026). Scale bars: 20 μm.The expression of 2 CBCstem cell markers Olfm4 and Lgr5 were examined by in situ hybridisation in control and Snai1KO small intestine. Lgr5 and Olfm4in situ hybridisation staining shows a loss of CBCstem cell marker expression after loss of Snai1 (high magnification inserts depict a representative crypt demarcated by a dotted line). Loss of Snai1 was confirmed in knockout tissue using immunohistochemistry. Scale bars: 20 μm.The analysis of sorted and EGFP‐positive cells from Lgr5‐EGFP‐IRES‐creERT2AhCreSnai1fl/fl and Lgr5‐EGFP‐IRES‐creERT2AhCreSnai1+/+mice 5 days after βNF induction show an ˜tenfold reduction in Lgr5GFP‐positive CBCstem cells.Immunohistochemistry for active caspase‐3 to detect apoptotic cells revealed a significant increase in the number of apoptotic cells in both crypt (n = 5, P = 0.0001) and villus (n = 5, P = 0.005) regions in Snai1KO tissue (arrows mark active caspase‐3‐positive cells). Scale bars: 20 μm.A lineage tracing strategy was employed to determine the fate of CBCcells that lack Snai1. Tissue from Lgr5‐EGFP‐IRES‐creERT2Snai1+/+ (control) and Lgr5‐EGFP‐IRES‐creERT2Snai1fl/fl (Snai1KO) that also contained the ROSA26LacZCre reporter allele was stained with X‐Gal 5 days after tamoxifen injection. Quantification of X‐Gal‐positive foci per cm shows Snai1 knockout tissue failed to lineage trace indicating that no progeny are produced from Snai1 knockout cells. Bars represent mean ± SD (n = 3, P = 0.0001). Scale bars: 0.50 mm. Two‐tailed Student's t‐test was used to assess significance. See also Supplementary Figure S2.
Figure 4VillinCreERT2Snai1fl/florganoids treated with tamoxifen show arrested growth after 2 and 4 days of treatment compared to controls. Scale bars: 50 μm.Quantification of viable cell growth in VillinCreERT2Snai1fl/florganoids treated with tamoxifen compared to controls over 4 days. Bars represent mean ± SD. n = 3 experiments per group, **P = 0.002.Assessment of apoptosis in control and VillinCreERT2 Snai1fl/fl organoids after 4 days of growth. Photographs show an overlay of bright‐field and fluorescent images with propidium iodide and Hoechst staining. Quantification of apoptotic organoids. Bars represent mean ± SD. n = 3 experiments per group, **P = 0.00001. Two‐tailed Student's t‐test was used to assess significance. Scale bars: 50 μm. See also Supplementary Figure S3.
Figure 5The consequences of Snai1 knockout were analysed over a time course. Immunohistochemistry for Snai1 revealed significant loss of expression at day 5 following induction compared to control. The epithelium is re‐populated by wild‐type cells as shown on day 60. Scale bars: 20 μm.
Figure 6A PAS staining was used to detect goblet cells in the small intestine, and no differences in cell numbers were observed in test versus control mice (n = 4, NS).B Immunohistochemistry for synaptophysin was used to detect enteroendocrine cells. Quantification of the number of enteroendocrine cells per crypt-villus axes revealed a significant decrease in enteroendocrine cell numbers when Snai1 levels are elevated (n = 4, P = 0.003).C Immunohistochemistry for lysozyme was used to detect Paneth cells. Quantification of Paneth cell numbers indicated a significant decrease in Paneth cells in the base of crypts in mice with elevated Snai1 levels (n = 5, P = 0.027).D, E Quantification of total PCNA‐positive cells per crypt revealed a significant increase when Snai1 levels were elevated (n = 4, P = 0.009). This was confirmed in VillinCreERT2 ROSA26Snai1 mice 5 days after induction with tamoxifen where analysis of (E) PCNA‐positive cells in the base of crypts revealed a significant increase in proliferating cells in crypts with elevated Snai1 levels compared to control tissue (n = 4, P = 0.0005). Bars represent mean ± SD.D, E Quantification of total PCNA‐positive cells per crypt revealed a significant increase when Snai1 levels were elevated (n = 4, P = 0.009). This was confirmed in VillinCreERT2 ROSA26Snai1 mice 5 days after induction with tamoxifen where analysis of (E) PCNA‐positive cells in the base of crypts revealed a significant increase in proliferating cells in crypts with elevated Snai1 levels compared to control tissue (n = 4, P = 0.0005). Bars represent mean ± SD.F PCNAimmunostaining (green) and DAPIstaining (blue) of VillinCreERT2ROSA26Snai1 compared to control VillinCreERT2 littermates. An expansion of proliferating CBCcells (indicated by arrows) is observed at the base of crypts. Scale bars: 10 μm.G qRT-PCR analysis of stem cell marker expression in the small intestine from VillinCreERT2 ROSA26Snai1 compared to control VillinCreER2T littermates. Bars represent mean ± SD. n = 3 experiments per group, Olfm4 P = 0.013, Cdh1/E‐cadherin P = 0.003, Lgr5 P = 0.013.H CD44v6 immunostaining of tissue from AhCre control compared to AhCre RosaSnai1 tissue. A significant increase in CD44v6‐positive cells localised at the base of crypts (depicted by arrows) was observed. Bars represent mean ± SD. n = 3 mice per group, *P = 0.00023. Two‐tailed Student's t‐test was used to assess significance. Scale bars: 10 μm.
Figure 7VillinCreERT2 RosaSnai1 organoids treated with tamoxifen show slower growth after 2 and 4 days of treatment compared to controls. Scale bars: 50 μm.Quantification of viable cells in VillinCreERT RosaSnai1 organoids treated with tamoxifen compared to control growth over 4 days. Bars represent mean ± SD. n = 3 experiments per group, **P = 0.002.Assessment of apoptosis in control and VillinCreERT2 RosaSnai1 organoids after 4 days of growth. Photographs show an overlay of bright‐field, propidium iodide and Hoechst fluorescence. Scale bars: 50 μm.Quantification of apoptotic organoids with and without Wnt3A treatment shown as mean ± SD. n = 3 experiments per group, **P = 0.00003. Two‐tailed Student's t‐test was used to assess significance.
Figure 8A-C Droplet digital PCR analysis of Bmf, Bbc3/Puma and Bcl2l 11/Bim after conditional Snai1 loss in in vitro organoid culture. VillinCreERT2 Snai1+/+ (control) and VillinCreERT2 Snai1fl/fl SI organoids were treated for 24 h with tamoxifen. No differences in gene expression were observed (n = 3).D qRT-PCR analysis of Serinc3 downregulation after conditional Snai1 loss in in vitro organoid culture. VillinCreERT2 Snai1+/+ (control) and VillinCreERT2 Snai1fl/fl SI organoids were treated for 24 (P = 0.0001) and 72 h (P = 0.006) with tamoxifen. Bars represent mean ± SD. n = 3 mice per group.E SW480 cells, which transiently express GFP‐SNAI1, were used to detect direct binding of SNAI1 to the promoters of known target genes CDH1, IL8 and a novel target SERINC3 revealed by microarray analysis. An unrelated DNA region was used as a negative control. Anti‐GFP antibody was used to immunoprecipitate GFP‐tagged SNAI1, while IgG control antibody was used as a negative control. The results are given as relative enrichment compared to input material and are representative of three independent experiments.
Figure 1Down regulation of SREBP target genes in Cideb-deficient liver. Microarray heatmap of fatty acids/cholesterol synthesis genes that were down-regulated in Cideb-/- mice and Scap liver-specific knockout mice, comparing to the respective WT controls (WT VS Cideb-/-, WT VS L-Scap-/-, n = 2 per group). The expression level of each gene was linearly normalized to range from -1 to 1 before plotting.Decreased SREBP processing and maturation in Cideb deficient liver. Immune blotting (IB) of the full length, precursor SREBP-1/2 (SREBP FL) and the cleaved, active SREBP-1/2 (SREBP N) from WT and Cideb-/- mice liver under ad-lib (ND), fasting 12h (F), and refeeding (R) conditions. Mice were refed with high-carbohydrate low-fat diet for 12h after 12h fasting.Defective SREBP transcriptional response to refeeding in Cideb-deficient liver. Transcript levels of lipogenic and cholesterol metabolism genes determined by qPCR from WT and Cideb-/- liver under ad-lib (ND), fasting 12h (F), and refeeding (R) conditions. Mice were refed with high-carbohydrate low-fat diet for 12h after 12h fasting (n = 3 per group).Re-introduction of Cideb rescues SREBP processing in Cideb-/- liver. WT and Cideb-/- mice were injected with AAV-GFP or AAV-Cideb, and sacrificed for IB with the indicated antibodies 20 days after the injection.Re-introduction of Cideb rescues the expression of SREBP target genes in Cideb-/- liver. Transcript levels of lipogenic genes determined by qPCR from WT and Cideb-/- liver. WT and Cideb-/- mice were injected with AAV-GFP or AAV-Cideb, and sacrificed for qPCR 20 days after the injection (n = 3 per group).