etv1 gene insert in plx trc311 etv1  (Addgene inc)

Journal: Nature Communications

Article Title: ETVs dictate hPSC differentiation by tuning biophysical properties

doi: 10.1038/s41467-025-56591-6

Figure Lengend Snippet: A Representative images of immunofluorescence staining show the ETV1 protein (green) presence in OCT3/4 (red) positive cells. DAPI was used to label nuclei (blue). Protein coexpression is presented on the right in enlarged inserts. N = 3 biological replicates. Scale bar = 100 μm. B Representative immunofluorescence staining shows the presence of ETV4 (red), and ETV5 (gray), in NANOG+ (green) cells. DAPI was used to label nuclei (blue). Protein coexpression is presented on the right in the enlarged insert. N = 3 biological replicates. Scale bar = 100 μm. C Representative flow cytometry analysis shows the coexpressions of: ETV1 and OCT3/4 proteins, and ETV4, ETV5, and KLF4 proteins in hPSCs; 97–99% of hPSCs expressed OCT3/4 and ETVs, while 99.4% and 99% of KLF4 + hPSCs expressed ETV4 and ETV5, respectively. N = 3 biological replicates. D Strategy to knock out ETV1 gene in hPSCs using CRISPR/Cas9 approach. Three different sgRNAs (red arrows) targeting exon 4 of the ETV1 gene were co-transfected into hPSCs, and pretreated with doxycycline to induce Cas9 expression, resulting in a 127-nucleotide deletion and a premature stop codon. Untranslated regions (gray), PEA3 domain (turquoise green), ETS domain (dark yellow), STOP codon (red). Primer positions are shown by arrowheads. E ETV1 protein absence in KO compared to WT hPSCs, demonstrated by western blotting. An antibody against GAPDH was used as a loading control. F The relative expression level of ETV4 and ETV5 was higher in ETV1 KO compared to WT cells (baseline set at 1), shown by qPCR. The data are presented as the means ± SDs. A two-sided student’s t -test was used to determine the p -values shown on the graph. N = 3 biological replicates. G , H Strategy to generate ETV4, ETV5 , and ETV1 triple KO (tKO) in hPSCs. Three sgRNAs (red arrows) targeting exons 2 and 3 of ETV4 ( G ), and exons 3 and 4 of ETV5 ( H ) were co-transfected into ETV1 KO hPSCs, leading to deletions and premature stop codons. Untranslated regions (gray), PEA3 domain (turquoise green), ETS domain (yellow), STOP codon (red). Primer positions are shown by arrowheads. I Western blot analysis of ETV4 or ETV5 proteins in tKO and WT hPSCs. An antibody against GAPDH was used as a loading control. N = 2 biological replicates. J Representative bright-field images of WT, KO, KO2, and tKO hPSC colonies. Scale bar = 200 μm. N = 5 biological replicates.

Article Snippet: The resulting ETV1 PCR product was isolated with the Monarch DNA Gel Extraction Kit (NEB, USA).

Techniques: Immunofluorescence, Staining, Flow Cytometry, Knock-Out, CRISPR, Transfection, Expressing, Western Blot, Control

Journal: Nature Communications

Article Title: ETVs dictate hPSC differentiation by tuning biophysical properties

doi: 10.1038/s41467-025-56591-6

Figure Lengend Snippet: A Growth dynamics of KO, tKO, and WT hPSCs over a 24 h culture monitored by live-cell imaging. Left - representative images of cell confluency at 2, 12, and 24 h culture, marked by green mask. Middle-representative images of cell number at 2, 12, and 24 h culture, marked by nuclei marker SiR DNA (red). Right - representative images of cell spreading (quantified as the ratio of confluency to cell number) at 2, 12, and 24 h culture. A green line marks confluency and nuclei are marked by SiR DNA in red. Scale bar = 400 µm. B Quantification of WT (yellow), KO (green), and tKO (maroon) hPSC confluency over a 24 h culture. tKO cells show the highest confluency, a 166% increase compared to WT cells. N = 3 biological replicates. At 24 h, for KO vs. WT and tKO vs. WT, p < 0.0001, for tKO vs. KO, p = 0.0025. C Quantification of cell number over a 24 h culture. KO (green) and tKO (maroon) show an increase in the cell number compared to WT cells (yellow). The increase in cell number was the most pronounced for tKO cells (66% increase in comparison to WT) and by 36% for KO hPSCs. N = 3 biological replicates. At 24 h, for KO vs. WT, p < 0.0001, for tKO vs. KO, p = 0.039; at 18 h, for tKO vs. WT, p = 0.0171. D Quantification of cell spreading (confluency/cell number) over a 24 h culture. KO (green) and tKO (maroon) show an increase in cell spreading compared to WT cells (yellow). N = 3 biological replicates. At 24 h, for KO vs. WT, p = 0.0008, for tKO vs. WT and tKO vs. KO, p < 0.0001. E Confluency quantification at 2 h post-seeding (corresponds to A ). The highest increase in cell confluency was noted for tKO followed by KO and KO2 in comparison to WT hPSCs. N = 3 biological replicates. For KO vs. WT, tKO vs. WT, tKO vs KO, p < 0.0001, for KO2 vs. WT, p = 0.0003. F Cell number quantification at 2 h post-seeding (corresponds to A ). The highest increase in cell number was noted for tKO followed by KO and KO2 hPSCs. N = 3 biological replicates. For KO vs. WT and tKO vs. WT, p < 0.0001, for KO2 vs. WT, p = 0.006, tKO vs. KO, p = 0.0006. G Quantification of cell spreading (confluency/cell number) at 2 h post-seeding (corresponds to A ). The most pronounced increase in cell spreading was noted for tKO hPSCs followed by KO and KO2. N = 3 biological replicates. For KO vs. WT, tKO vs. WT, tKO vs KO, p < 0.0001, for KO2 vs. WT, p = 0.0009. H Representative crystal violet staining images of cells cultured in the absence (top panel) or presence (bottom panel) of ROCK inhibitor (ROCKi) 24 h after hPSC seeding. The same number of WT and KO cells were seeded on different surface coatings, as indicated. Scale bar = 400 μm. I Quantification of crystal violet staining shows an increase in the attachment of KO (green) and tKO (maroon) compared to WT (yellow) cells, on all tested surface coatings in the absence (top panel) and presence (bottom panel) of ROCKi. N = 4 biological replicates. J Physical cytometer analysis of KO, tKO, and WT hPSC spheres demonstrated enhanced density of KO and tKO compared to WT. N = 3 biological replicates. K Representative images of immunofluorescence staining show ETV1 (green) overexpression in hPSCs induced by 24 h doxycycline treatment (Dox+), compared to untreated (Dox-) cells. L Quantification of adhesion protein levels CDH1 (left) and ITGA5 (right) in WT_OE and KO_OE hPSCs after induction of ETV1 overexpression. CDH1, N = 4 biological replicates; ITGA5, N = 3 biological replicates. For plots B–G, I, J , and L , a one-way ANOVA for multiple comparisons was used to determine the p -values shown on the graph. The data are presented as means ± SDs.

Article Snippet: The resulting ETV1 PCR product was isolated with the Monarch DNA Gel Extraction Kit (NEB, USA).

Techniques: Live Cell Imaging, Marker, Comparison, Staining, Cell Culture, Cytometry, Immunofluorescence, Over Expression

Journal: Nature Communications

Article Title: ETVs dictate hPSC differentiation by tuning biophysical properties

doi: 10.1038/s41467-025-56591-6

Figure Lengend Snippet: A A volcano plot was generated to visualize differentially expressed genes between KO and WT hPSCs. Red dots indicate 133 upregulated genes, and blue dots indicate 95 downregulated genes, showing log2 fold change (log2 FC) and −log10 adjusted p -value (−log10 adjp). B A volcano plot was generated to visualize differentially expressed genes between tKO and WT hPSCs. Red dots indicate 1201 upregulated genes, and blue dots indicate 2027 downregulated genes, showing log2 fold change (log2 FC) and −log10 adjusted p- value (−log10 adjp). C Volcano plot of differentially expressed genes between tKO and KO hPSCs shows log2 fold change (log2 FC) and −log10 adjusted p- value (−log10 adjp) for 679 downregulated (blue) and 400 upregulated (red) genes. D The top-ranked enriched terms (adjusted p-value ≤ 0.05) from the KEGG, WikiPathways, and Biological Processes databases were identified among the differentially expressed genes between KO and WT hPSCs. Enriched functional terms associated with upregulated and downregulated genes in KO compared to WT hPSCs are marked by red and blue triangles, respectively. E The top-ranked enriched terms (adjusted p-value ≤ 0.05) from the KEGG, WikiPathways, and Biological Processes databases among the differentially expressed genes between tKO and WT hPSCs. Enriched functional terms associated with upregulated and downregulated genes in tKO compared to WT hPSCs are marked by red and blue triangles, respectively. F The top-ranked enriched terms (adjusted p-value ≤ 0.05) from the KEGG, WikiPathways, and Biological Processes databases among differentially expressed genes between tKO and KO hPSCs. Enriched functional terms associated with upregulated and downregulated genes in tKO compared to WT hPSCs are marked by red and blue triangles, respectively. G Heatmaps demonstrate the top significant differentially expressed genes in KO compared to WT associated with cell–cell and cell-ECM adhesion, PI3K/AKT pathway (left panel), or pluripotency and cell fate (right panel). Diff. – differentiation. H Heatmaps demonstrate the top significant differentially expressed genes in tKO compared to WT associated with cell–cell and cell-ECM adhesion, and PI3K/AKT pathway (left panel) or pluripotency and cell fate (right panel). Diff. – differentiation. I ATAC-seq tracks highlight the ITGAX locus in WT (yellow), KO (green), and tKO (maroon) hPSCs. The peaks represent normalized and combined biological replicates ( N = 2). J ATAC-seq tracks highlight the VAV1 locus in WT (yellow), KO (green), and tKO (maroon) hPSCs. The peaks represent normalized and combined biological replicates ( N = 2). K Western blot demonstrates the ETV1 presence in both the chromatin-bound (CH) and soluble protein fraction (SF) in WT hPSCs. pHH3 was used as a positive control of fractionation, and total cell lysate (TL) was used as the positive control for ETV1. N = 2 biological replicates. L ChIP-qPCR analysis (antibody against ETV1) confirmed ETV1 binding to the promoter region of selected differentially expressed genes connected to cell adhesion, i.e., ITGA5 , VCL , PDGFRB , and COL4A or pluripotency, i.e., JUN and CDKN1A . IgG – control (blue); ChIP ETV1 – target (red). Data are presented as the mean ± SDs. N = 2 biological replicates. For plots ( A – C ), the DESeq2 package and the Wald test were used to determine significance. DAVID online software was used for biological term enrichment analysis of the DEGs, with the Fisher Exact statistics to list annotation terms and their associated genes. The p -values are adjusted for multiple comparisons using the Benjamini and Hochberg approach. Genes with non-significant change in expression are depicted in gray (−log10 (adjp) <2; log2 FC between 0.5 and −0.5). Highlighted dots indicate selected genes in the enriched pathways, pictured in plots ( D – F ).

Article Snippet: The resulting ETV1 PCR product was isolated with the Monarch DNA Gel Extraction Kit (NEB, USA).

Techniques: Generated, Functional Assay, Western Blot, Positive Control, Fractionation, ChIP-qPCR, Binding Assay, Control, Software, Expressing

Journal: Nature Communications

Article Title: ETVs dictate hPSC differentiation by tuning biophysical properties

doi: 10.1038/s41467-025-56591-6

Figure Lengend Snippet: A Representative images of immunofluorescence staining of ITGA5 (red) protein in WT and KO hPSCs. DAPI marks the nuclei (blue). The zoomed inset highlights a selected area from the merged image. Scale bar = 50 μm. B Representative images of immunofluorescence staining of VCL (red) protein in WT and KO hPSCs. DAPI marks the nuclei (blue). The zoomed inset highlights a selected area from the merged image. Scale bar = 50 μm. C Western blot analysis of VCL protein expression in WT, KO, and tKO hPSCs. An antibody against GAPDH serves as a loading control. N = 3 biological replicates. D Representative images of immunofluorescence staining of CDH1 (green) and PXN (red) protein localization in WT, KO, and tKO hPSCs. CDH1 and PXN distribution within colonies is changed in KO and tKO compared to WT, and in KO compared to tKO. DAPI marks the nuclei (blue). The zoomed inset highlights a selected area from the merged image. Scale bar = 50 μm. E High-resolution SIM microscopy images (3D projections) of CDH1 (upper panel) and PXN (bottom panel) immunofluorescence staining in WT, KO, and tKO hPSC colonies. F Representative images of CDH1 (green) show disrupted colony integrity in tKO. N = 3 biological replicates. Scale bar = 100 μm. G High-resolution microscopy images of F-ACTIN (green) immunofluorescence staining show different actin cytoskeleton organization at the colony edges of WT and tKO hPSC. Scale bar = 20 nm. H Western blotting analysis of phospho-AKT (p-AKT), total AKT protein level in WT, KO, and tKO hPSCs. An antibody against GAPDH was used as a loading control. N = 3 biological replicates. I Representative bright-field images of WT and KO hPSCs (cells marked by a yellow mask) after 24 h treatment with PI3K/AKT pathway inhibitors (PI-103, Torin2) or an activator (insulin). Untreated and DMSO-treated WT and KO hPSCs served as controls. Scale bar = 400 μm. J Quantification of crystal violet staining shows a dose-dependent decrease in the attachment of KO (green) after PI3K/AKT pathway inhibition by PI-103, compared to untreated (UT) and control (DMSO) KO hPSCs. N = 4 biological replicates. KO: UT vs. 5 nM, p = 0.0003, UT vs. 15 nM and UT vs. 30 nM, p < 0.0001; 30 nM KO vs. UT WT, p = 0.0422. K Quantification of crystal violet staining shows a dose-dependent decrease in cell attachment following Torin2 treatment, compared to both untreated (UT) and DMSO-treated KO hPSCs. Treatment with 15 nM Torin2 resulted in a proportion of attached KO cells similar to untreated WT (yellow) hPSCs. Treatment with 200 nM Torin2 resulted in decreased attachment of KO compared to WT hPSCs. N = 4 biological replicates. KO: UT vs. 2 nM, p = 0.0003, UT vs. 15 nM, and UT vs. 200 nM, p < 0.0001; 15 nM KO vs. UT WT, p = ns. L Quantification of crystal violet staining shows a dose-dependent increase of WT (yellow) attachment after insulin treatment, compared to untreated (UT) WT hPSCs. The 3 µg/ml insulin treatment resulted in a similar number of attached WT cells as in untreated (UT) KO hPSCs (WT: UT vs. 3 µg/ml, p < 0.0001; KO: UT vs. 3 µg/ml, p = ns). The fraction of KO (green) hPSCs did not change following insulin treatment compared to UT KO hPSCs (all doses, p = ns). No difference in the number of attached KO hPSCs was observed regardless of the insulin dose, indicating that ETV1 deletion may cause saturation of PI3K/AKT pathway activity. N = 4 biological replicates. M Representative images of immunofluorescence of CDH1 (green) and PXN (red) proteins in Torin2 or insulin-treated WT and KO hPSCs. DAPI marks the nuclei in blue. Scale bar = 200 μm. N Quantification of fluorescence intensity (CDH1 + /DAPI) from immunofluorescence images of untreated (UT), DMSO-, Torin2- (TORIN2), and insulin-treated (INS) WT (yellow) and KO (green) hPSCs. N = 3 biological replicates. WT: UT vs. DMSO, p = ns, UT vs. TORIN2, p = 0.0016, UT vs. INS, p = 0.0447; KO: UT vs. DMSO and UT vs. INS, p = ns, UT vs. TORIN2, p = 0.0001. N Quantification of fluorescence intensity (PXN + /DAPI) from immunofluorescence images of untreated (UT), DMSO-, Torin2- (TORIN2) or insulin-treated (INS) WT (yellow) and KO (green) hPSCs. N = 3 biological replicates. WT: UT vs. DMSO, p = ns, UT vs. TORIN2 and UT vs. INS, p < 0.0001; KO: UT vs. DMSO and UT vs. INS, p = ns, UT vs. TORIN2, p < 0.0001. For plots ( J – L, N , and O ), a one-way ANOVA for multiple comparisons was used to determine the p -values shown on the graph. The data are presented as means ± SDs.

Article Snippet: The resulting ETV1 PCR product was isolated with the Monarch DNA Gel Extraction Kit (NEB, USA).

Techniques: Immunofluorescence, Staining, Western Blot, Expressing, Control, Microscopy, Inhibition, Cell Attachment Assay, Activity Assay, Fluorescence

Journal: Nature Communications

Article Title: ETVs dictate hPSC differentiation by tuning biophysical properties

doi: 10.1038/s41467-025-56591-6

Figure Lengend Snippet: A Scheme illustrating the micropatterning approach to in vitro generation of the organized embryo-like structures (gastruloids). hPSCs (light blue) were plated on laminin 521-coated micropatterns and exposed for 46 h to BMP4 to induce self-organization and formation of gastruloids, containing extraembryonic-like cells (red), ectodermal cells (green) and the mesodermal and endodermal cells (dark blue). B Representative immunofluorescence staining of ISL1 (red), SOX2 (green), and BRA (blue) in gastruloids generated from ESI (hPSC control cell line), HUES8 (hPSC control cell line), iCas9 (WT hPSCs, parental control), KO and tKO hPSCs. Note, the disrupted germ layers organization in KO hPSCs (SOX2+ and BRA+), the overgrowth of extraembryonic-like cells (ISL1+), and the absence of SOX2+ cells in tKO, compared to control hPSC lines (ESI, HUES8 and iCas9). Scale bars = 200 µm. C Distribution from the center to the gastruloid edge of ISL1, SOX2, and BRA positive signals based on immunofluorescence staining on ESI (blue), HUES8 (red), iCas9 (green), tKO (black), and KO (pink) gastruloids. Data are presented as the mean ± SEM. N = 3 biological replicates. D WT, KO, and tKO differentiation to definitive endoderm using a standard protocol with full doses of Activin A and CHIR99021 (a WNT activator). N = 3 biological replicates. KO vs. WT, p = ns, tKO vs. WT, p = 0.0232, tKO vs. KO, p = 0.0002. E WT, KO, and tKO differentiation to definitive endoderm using a suboptimal protocol with decreased concertation of Activin A, with or without CHIR99021. N = 3 biological replicates. KO vs. WT and tKO vs. KO, p < 0.0001, tKO vs. WT, p = ns. F Scheme illustrating hPSC spontaneous differentiation into embryoid bodies (EBs). G Gene expression changes in KO EBs reveal altered differentiation potential. Dot plot shows log2FC in gene expression (qPCR) between KO and WT EBs at days 8 and 10 of spontaneous differentiation. The increased expression of endodermal and mesodermal markers and decreased expression of ectodermal markers were observed in KO at both timepoints. N = 3 biological replicates. The p -values were determined by two-sided student’s t -test. H Representative immunofluorescence images of human fetal pancreas at weeks 10.6 and 13 of embryogenesis stained with PDX1 (red), CHGA (gray), and ETV1 (green). N = 3 tissue samples. Scale bar = 100 µm. I Representative immunofluorescence staining of ETV1 (green) in WT hPSC-derived definitive endoderm (DE, upper panel), and pancreatic progenitors (PP, bottom panel). SOX17 (red) marks DE, and PDX1 (green) and NKX6-1 (red) mark PPs. N = 3 biological replicates. Scale bar = 200 µm. J Scheme illustrating hPSC differentiation into pancreatic progenitors (PP). During differentiation, hPSCs pass through the following stages: definitive endoderm (DE) marked by FOXA2 and SOX17 expression, PP marked by PDX1 and NKX6-1 expression. At the PP stage (day 12) single-cell RNA sequencing and ATAC sequencing were performed on WT and KO samples. K Representative immunofluorescence staining of PDX1 (green), NKX6-1 (red), and CHGA (gray) proteins in WT and KO PPs. N = 3 biological replicates. Scale bar = 200 µm. L Representative flow cytometry analysis showing the absence of NKX6-1+KO PPs and decreased PDX1 protein levels in KO compared to WT PPs. Two distinct PDX1+ populations are observed in KO cells: low PDX1+ (~50% of cells) and high PDX1+ (~50% of cells). In contrast, WT PPs exhibit predominantly high PDX1 expression. For plots ( D , E ), a one-way ANOVA with Turkey’s correction for multiple comparisons was used to determine the p -values shown on the graph. The data are presented as means ± SDs.

Article Snippet: The resulting ETV1 PCR product was isolated with the Monarch DNA Gel Extraction Kit (NEB, USA).

Techniques: In Vitro, Immunofluorescence, Staining, Generated, Control, Gene Expression, Expressing, Derivative Assay, RNA Sequencing, Sequencing, Flow Cytometry

Journal: Disease Models & Mechanisms

Article Title: ETV1 is a key regulator of enteroendocrine PYY production

doi: 10.1242/dmm.052610

Figure Lengend Snippet: Etv1 expression is most strongly correlated with expression of Gcg , Pyy and Cck in vivo . (A) Workflow for analysis of enteroendocrine cells (EECs) from the single-cell RNA-sequencing (scRNA-seq) dataset published by . PCA, principal component analysis; UMAP, Uniform Manifold Approximation and Projection. (B) Expression of Etv1 across different cell type clusters (original cell type annotation). TA, transit amplifying. (C) UMAP plot following unsupervised clustering of EECs from mouse small intestine. Clusters are annotated based on expression of known marker genes with a temporal expression pattern during EEC differentiation. EC, enterochromaffin cell. (D) Violin plots showing expression of selected genes involved in EEC differentiation across cell type clusters of EECs from mouse small intestine. (E) UMAP plot showing expression levels of Etv1 in EECs from mouse small intestine. (F) Correlation between Etv1 expression and expression of different enteroendocrine (EE) hormones. R-value=Pearson correlation coefficient. (G-I) UMAP plots showing expression of Gcg (G), Pyy (H) and Cck (I) in EECs from mouse small intestine.

Article Snippet: The Etv1 gene insert in pLX_TRC311_ETV1 ( Addgene plasmid #74981 ) was cloned into SP170 (PB-TRE-DEST-IRES-BSD) (a kind gift from Steve Pollard's laboratory, Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK) using gateway cloning.

Techniques: Expressing, In Vivo, RNA Sequencing, Marker

Journal: Disease Models & Mechanisms

Article Title: ETV1 is a key regulator of enteroendocrine PYY production

doi: 10.1242/dmm.052610

Figure Lengend Snippet: EEC differentiation and Etv1 expression in organoid cultures resemble in vivo observations. (A) Expression of Etv1 across different cell type clusters (cell type annotation from ). (B) UMAP plot following unsupervised clustering of EECs from organoid cultures. Clusters are annotated based on expression of known marker genes with a temporal expression pattern during EEC differentiation. (C) Violin plots showing expression of selected transcription factors involved in EEC differentiation across cell type clusters of EECs in organoids. (D) UMAP plot showing expression levels of Etv1 in EECs from organoids. (E) Correlation between Etv1 expression and expression of different EE hormones in organoid cultures. R-value=Pearson correlation coefficient.

Article Snippet: The Etv1 gene insert in pLX_TRC311_ETV1 ( Addgene plasmid #74981 ) was cloned into SP170 (PB-TRE-DEST-IRES-BSD) (a kind gift from Steve Pollard's laboratory, Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK) using gateway cloning.

Techniques: Expressing, In Vivo, Marker

Journal: Disease Models & Mechanisms

Article Title: ETV1 is a key regulator of enteroendocrine PYY production

doi: 10.1242/dmm.052610

Figure Lengend Snippet: Etv1 mutant cultures have reduced Pyy expression. (A) Strategy for generation of Etv1 mutant organoid lines. Ngn3, Neurog3 . Created in BioRender by Jensen Team (2025). https://BioRender.com/eqtfeop . This figure was sublicensed under CC-BY 4.0 terms. (B) Amplified and sequenced Etv1 cDNA aligned to the Etv1 gene using the BLAT alignment tool. Screenshot downloaded from http://genome.ucsc.edu . (C) ETV1 protein (transcript variant 1). One dot corresponds to one amino acid (AA). Skipping of exon 8 changes AA 186-187 from phenylalanine (F) and arginine (R) to serine (S) and alanine (A) and introduces a premature stop codon after AA187, resulting in a protein that lacks the DNA binding domain (orange dots). (D-G) Expression of Etv1 (D), Gcg (E), Cck (F) and Pyy (G) in control and Etv1 mutant organoid cultures. The Etv1 reverse primer is located within exon 8. Expression is normalised to expression of Gapdh. Error bars indicate s.d. ( n =3). Significance was evaluated with an unpaired two-tailed t -test. CTRL, control. (H,I) Percentage of Neurog3-RFP + (H) and Gcg-Venus + (I) cells in control (two lines) and Etv1 mutant (three lines) organoid cultures assessed by flow cytometry. Error bars indicate s.d. Significance was evaluated with an unpaired two-tailed t -test. (J,K) Expression of Etv1 (J) and Pyy (K) in control and Etv1 mutant organoid cultures treated for 3 days with or without 10 µg DAPT and/or 20 ng/ml BMP-4. Significance was evaluated with an unpaired two-tailed t -test. ns, not significant; * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001.

Article Snippet: The Etv1 gene insert in pLX_TRC311_ETV1 ( Addgene plasmid #74981 ) was cloned into SP170 (PB-TRE-DEST-IRES-BSD) (a kind gift from Steve Pollard's laboratory, Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK) using gateway cloning.

Techniques: Mutagenesis, Expressing, Amplification, Variant Assay, Binding Assay, Control, Two Tailed Test, Flow Cytometry

Journal: Disease Models & Mechanisms

Article Title: ETV1 is a key regulator of enteroendocrine PYY production

doi: 10.1242/dmm.052610

Figure Lengend Snippet: Etv1 mutant organoids show no overall changes in cell type composition but lack EECs with high Pyy expression. (A) UMAP plot of cells from both control (two lines) and Etv1 mutant (three lines) organoids following scRNA-seq (1158 cells in total). Cell types are annotated based on expression of known marker genes ( Fig. S4A ). (B) UMAP plot of cells from control (left) and Etv1 mutant (right) organoids (control, 494 cells; Etv1 mutant, 664 cells). (C) Percentage of cells found in each of the identified cell clusters in control (two lines) and Etv1 mutant (three lines) organoids. Error bars indicate s.d. Significance was evaluated with an unpaired two-tailed t -test. ns, not significant. (D) Violin plots showing expression levels of known cell type and proliferation marker genes in control (orange) and Etv1 mutant (green) organoids. (E) UMAP plot showing EECs in control (orange) and Etv1 mutant (green) organoids. (F) UMAP plot showing expression levels of Etv1 , Gcg , Cck and Pyy in EECs from control (top row) and Etv1 mutant (bottom row) organoids.

Article Snippet: The Etv1 gene insert in pLX_TRC311_ETV1 ( Addgene plasmid #74981 ) was cloned into SP170 (PB-TRE-DEST-IRES-BSD) (a kind gift from Steve Pollard's laboratory, Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK) using gateway cloning.

Techniques: Mutagenesis, Expressing, Control, Marker, Two Tailed Test

Journal: Disease Models & Mechanisms

Article Title: ETV1 is a key regulator of enteroendocrine PYY production

doi: 10.1242/dmm.052610

Figure Lengend Snippet: Etv1 overexpression increases expression of Pyy and Cck , but not Gcg . (A) Strategy for generation of Etv1 -overexpressing ( Etv1 OE) organoids. SI, small intestine. Created in BioRender by Jensen Team (2025). https://BioRender.com/eqtfeop . This figure was sublicensed under CC-BY 4.0 terms. (B) Images of control and Etv1 OE organoids with and without 48 h of doxycycline treatment. Scale bars: 275 µm. Organoids were derived from a Neurog3 -RFP; Gcg -Venus mouse ( ; ). (C) Expression of Etv1 , Pyy , Gcg , Cck , Sct and Ngn3 ( Neurog3 ) in control and Etv1 OE organoid cultures with and without 48 h doxycycline treatment. Error bars indicate s.d. ( n =3). Expression is normalised to expression of 36B4 ( Rplp0 ). Significance was evaluated with a one-way ANOVA. (D) Luciferase activity in inducible Etv1 OE HEK293 cells transfected with a pGL4.23 vector containing either a wild-type (PyyProm_WT) or mutated (PyyProm_MUT) version of a 517 bp region upstream of Pyy covering two putative ETV1 binding sites ( Fig. S7A ). Luciferase activity was normalised to the activity in HEK293 cells transfected with a pGL4.23 vector without any insert. Where indicated, cells were treated for 24 h with doxycycline (1 mg/ml). Error bars indicate s.d. ( n =4). Significance was evaluated with an unpaired two-tailed t -test. ns, not significant; * P <0.05, ** P <0.01.

Article Snippet: The Etv1 gene insert in pLX_TRC311_ETV1 ( Addgene plasmid #74981 ) was cloned into SP170 (PB-TRE-DEST-IRES-BSD) (a kind gift from Steve Pollard's laboratory, Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK) using gateway cloning.

Techniques: Over Expression, Expressing, Control, Derivative Assay, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Binding Assay, Two Tailed Test

Journal: Nature Communications

Article Title: ETVs dictate hPSC differentiation by tuning biophysical properties

doi: 10.1038/s41467-025-56591-6

Figure Lengend Snippet: A Representative images of immunofluorescence staining show the ETV1 protein (green) presence in OCT3/4 (red) positive cells. DAPI was used to label nuclei (blue). Protein coexpression is presented on the right in enlarged inserts. N = 3 biological replicates. Scale bar = 100 μm. B Representative immunofluorescence staining shows the presence of ETV4 (red), and ETV5 (gray), in NANOG+ (green) cells. DAPI was used to label nuclei (blue). Protein coexpression is presented on the right in the enlarged insert. N = 3 biological replicates. Scale bar = 100 μm. C Representative flow cytometry analysis shows the coexpressions of: ETV1 and OCT3/4 proteins, and ETV4, ETV5, and KLF4 proteins in hPSCs; 97–99% of hPSCs expressed OCT3/4 and ETVs, while 99.4% and 99% of KLF4 + hPSCs expressed ETV4 and ETV5, respectively. N = 3 biological replicates. D Strategy to knock out ETV1 gene in hPSCs using CRISPR/Cas9 approach. Three different sgRNAs (red arrows) targeting exon 4 of the ETV1 gene were co-transfected into hPSCs, and pretreated with doxycycline to induce Cas9 expression, resulting in a 127-nucleotide deletion and a premature stop codon. Untranslated regions (gray), PEA3 domain (turquoise green), ETS domain (dark yellow), STOP codon (red). Primer positions are shown by arrowheads. E ETV1 protein absence in KO compared to WT hPSCs, demonstrated by western blotting. An antibody against GAPDH was used as a loading control. F The relative expression level of ETV4 and ETV5 was higher in ETV1 KO compared to WT cells (baseline set at 1), shown by qPCR. The data are presented as the means ± SDs. A two-sided student’s t -test was used to determine the p -values shown on the graph. N = 3 biological replicates. G , H Strategy to generate ETV4, ETV5 , and ETV1 triple KO (tKO) in hPSCs. Three sgRNAs (red arrows) targeting exons 2 and 3 of ETV4 ( G ), and exons 3 and 4 of ETV5 ( H ) were co-transfected into ETV1 KO hPSCs, leading to deletions and premature stop codons. Untranslated regions (gray), PEA3 domain (turquoise green), ETS domain (yellow), STOP codon (red). Primer positions are shown by arrowheads. I Western blot analysis of ETV4 or ETV5 proteins in tKO and WT hPSCs. An antibody against GAPDH was used as a loading control. N = 2 biological replicates. J Representative bright-field images of WT, KO, KO2, and tKO hPSC colonies. Scale bar = 200 μm. N = 5 biological replicates.

Article Snippet: The supernatant was immunoprecipitated overnight at 4 °C with either anti-ETV1 antibody (20 μg; Thermo Fisher Scientific, cat. number: PA5-67447) or control IgG antibody (1 μg; Thermo Fisher Scientific, cat. number: 02-6102) using magnetic protein A/G beads.

Techniques: Immunofluorescence, Staining, Flow Cytometry, Knock-Out, CRISPR, Transfection, Expressing, Western Blot, Control

Journal: Nature Communications

Article Title: ETVs dictate hPSC differentiation by tuning biophysical properties

doi: 10.1038/s41467-025-56591-6

Figure Lengend Snippet: A Growth dynamics of KO, tKO, and WT hPSCs over a 24 h culture monitored by live-cell imaging. Left - representative images of cell confluency at 2, 12, and 24 h culture, marked by green mask. Middle-representative images of cell number at 2, 12, and 24 h culture, marked by nuclei marker SiR DNA (red). Right - representative images of cell spreading (quantified as the ratio of confluency to cell number) at 2, 12, and 24 h culture. A green line marks confluency and nuclei are marked by SiR DNA in red. Scale bar = 400 µm. B Quantification of WT (yellow), KO (green), and tKO (maroon) hPSC confluency over a 24 h culture. tKO cells show the highest confluency, a 166% increase compared to WT cells. N = 3 biological replicates. At 24 h, for KO vs. WT and tKO vs. WT, p < 0.0001, for tKO vs. KO, p = 0.0025. C Quantification of cell number over a 24 h culture. KO (green) and tKO (maroon) show an increase in the cell number compared to WT cells (yellow). The increase in cell number was the most pronounced for tKO cells (66% increase in comparison to WT) and by 36% for KO hPSCs. N = 3 biological replicates. At 24 h, for KO vs. WT, p < 0.0001, for tKO vs. KO, p = 0.039; at 18 h, for tKO vs. WT, p = 0.0171. D Quantification of cell spreading (confluency/cell number) over a 24 h culture. KO (green) and tKO (maroon) show an increase in cell spreading compared to WT cells (yellow). N = 3 biological replicates. At 24 h, for KO vs. WT, p = 0.0008, for tKO vs. WT and tKO vs. KO, p < 0.0001. E Confluency quantification at 2 h post-seeding (corresponds to A ). The highest increase in cell confluency was noted for tKO followed by KO and KO2 in comparison to WT hPSCs. N = 3 biological replicates. For KO vs. WT, tKO vs. WT, tKO vs KO, p < 0.0001, for KO2 vs. WT, p = 0.0003. F Cell number quantification at 2 h post-seeding (corresponds to A ). The highest increase in cell number was noted for tKO followed by KO and KO2 hPSCs. N = 3 biological replicates. For KO vs. WT and tKO vs. WT, p < 0.0001, for KO2 vs. WT, p = 0.006, tKO vs. KO, p = 0.0006. G Quantification of cell spreading (confluency/cell number) at 2 h post-seeding (corresponds to A ). The most pronounced increase in cell spreading was noted for tKO hPSCs followed by KO and KO2. N = 3 biological replicates. For KO vs. WT, tKO vs. WT, tKO vs KO, p < 0.0001, for KO2 vs. WT, p = 0.0009. H Representative crystal violet staining images of cells cultured in the absence (top panel) or presence (bottom panel) of ROCK inhibitor (ROCKi) 24 h after hPSC seeding. The same number of WT and KO cells were seeded on different surface coatings, as indicated. Scale bar = 400 μm. I Quantification of crystal violet staining shows an increase in the attachment of KO (green) and tKO (maroon) compared to WT (yellow) cells, on all tested surface coatings in the absence (top panel) and presence (bottom panel) of ROCKi. N = 4 biological replicates. J Physical cytometer analysis of KO, tKO, and WT hPSC spheres demonstrated enhanced density of KO and tKO compared to WT. N = 3 biological replicates. K Representative images of immunofluorescence staining show ETV1 (green) overexpression in hPSCs induced by 24 h doxycycline treatment (Dox+), compared to untreated (Dox-) cells. L Quantification of adhesion protein levels CDH1 (left) and ITGA5 (right) in WT_OE and KO_OE hPSCs after induction of ETV1 overexpression. CDH1, N = 4 biological replicates; ITGA5, N = 3 biological replicates. For plots B–G, I, J , and L , a one-way ANOVA for multiple comparisons was used to determine the p -values shown on the graph. The data are presented as means ± SDs.

Article Snippet: The supernatant was immunoprecipitated overnight at 4 °C with either anti-ETV1 antibody (20 μg; Thermo Fisher Scientific, cat. number: PA5-67447) or control IgG antibody (1 μg; Thermo Fisher Scientific, cat. number: 02-6102) using magnetic protein A/G beads.

Techniques: Live Cell Imaging, Marker, Comparison, Staining, Cell Culture, Cytometry, Immunofluorescence, Over Expression

Journal: Nature Communications

Article Title: ETVs dictate hPSC differentiation by tuning biophysical properties

doi: 10.1038/s41467-025-56591-6

Figure Lengend Snippet: A A volcano plot was generated to visualize differentially expressed genes between KO and WT hPSCs. Red dots indicate 133 upregulated genes, and blue dots indicate 95 downregulated genes, showing log2 fold change (log2 FC) and −log10 adjusted p -value (−log10 adjp). B A volcano plot was generated to visualize differentially expressed genes between tKO and WT hPSCs. Red dots indicate 1201 upregulated genes, and blue dots indicate 2027 downregulated genes, showing log2 fold change (log2 FC) and −log10 adjusted p- value (−log10 adjp). C Volcano plot of differentially expressed genes between tKO and KO hPSCs shows log2 fold change (log2 FC) and −log10 adjusted p- value (−log10 adjp) for 679 downregulated (blue) and 400 upregulated (red) genes. D The top-ranked enriched terms (adjusted p-value ≤ 0.05) from the KEGG, WikiPathways, and Biological Processes databases were identified among the differentially expressed genes between KO and WT hPSCs. Enriched functional terms associated with upregulated and downregulated genes in KO compared to WT hPSCs are marked by red and blue triangles, respectively. E The top-ranked enriched terms (adjusted p-value ≤ 0.05) from the KEGG, WikiPathways, and Biological Processes databases among the differentially expressed genes between tKO and WT hPSCs. Enriched functional terms associated with upregulated and downregulated genes in tKO compared to WT hPSCs are marked by red and blue triangles, respectively. F The top-ranked enriched terms (adjusted p-value ≤ 0.05) from the KEGG, WikiPathways, and Biological Processes databases among differentially expressed genes between tKO and KO hPSCs. Enriched functional terms associated with upregulated and downregulated genes in tKO compared to WT hPSCs are marked by red and blue triangles, respectively. G Heatmaps demonstrate the top significant differentially expressed genes in KO compared to WT associated with cell–cell and cell-ECM adhesion, PI3K/AKT pathway (left panel), or pluripotency and cell fate (right panel). Diff. – differentiation. H Heatmaps demonstrate the top significant differentially expressed genes in tKO compared to WT associated with cell–cell and cell-ECM adhesion, and PI3K/AKT pathway (left panel) or pluripotency and cell fate (right panel). Diff. – differentiation. I ATAC-seq tracks highlight the ITGAX locus in WT (yellow), KO (green), and tKO (maroon) hPSCs. The peaks represent normalized and combined biological replicates ( N = 2). J ATAC-seq tracks highlight the VAV1 locus in WT (yellow), KO (green), and tKO (maroon) hPSCs. The peaks represent normalized and combined biological replicates ( N = 2). K Western blot demonstrates the ETV1 presence in both the chromatin-bound (CH) and soluble protein fraction (SF) in WT hPSCs. pHH3 was used as a positive control of fractionation, and total cell lysate (TL) was used as the positive control for ETV1. N = 2 biological replicates. L ChIP-qPCR analysis (antibody against ETV1) confirmed ETV1 binding to the promoter region of selected differentially expressed genes connected to cell adhesion, i.e., ITGA5 , VCL , PDGFRB , and COL4A or pluripotency, i.e., JUN and CDKN1A . IgG – control (blue); ChIP ETV1 – target (red). Data are presented as the mean ± SDs. N = 2 biological replicates. For plots ( A – C ), the DESeq2 package and the Wald test were used to determine significance. DAVID online software was used for biological term enrichment analysis of the DEGs, with the Fisher Exact statistics to list annotation terms and their associated genes. The p -values are adjusted for multiple comparisons using the Benjamini and Hochberg approach. Genes with non-significant change in expression are depicted in gray (−log10 (adjp) <2; log2 FC between 0.5 and −0.5). Highlighted dots indicate selected genes in the enriched pathways, pictured in plots ( D – F ).

Article Snippet: The supernatant was immunoprecipitated overnight at 4 °C with either anti-ETV1 antibody (20 μg; Thermo Fisher Scientific, cat. number: PA5-67447) or control IgG antibody (1 μg; Thermo Fisher Scientific, cat. number: 02-6102) using magnetic protein A/G beads.

Techniques: Generated, Functional Assay, Western Blot, Positive Control, Fractionation, ChIP-qPCR, Binding Assay, Control, Software, Expressing

Journal: Nature Communications

Article Title: ETVs dictate hPSC differentiation by tuning biophysical properties

doi: 10.1038/s41467-025-56591-6

Figure Lengend Snippet: A Representative images of immunofluorescence staining of ITGA5 (red) protein in WT and KO hPSCs. DAPI marks the nuclei (blue). The zoomed inset highlights a selected area from the merged image. Scale bar = 50 μm. B Representative images of immunofluorescence staining of VCL (red) protein in WT and KO hPSCs. DAPI marks the nuclei (blue). The zoomed inset highlights a selected area from the merged image. Scale bar = 50 μm. C Western blot analysis of VCL protein expression in WT, KO, and tKO hPSCs. An antibody against GAPDH serves as a loading control. N = 3 biological replicates. D Representative images of immunofluorescence staining of CDH1 (green) and PXN (red) protein localization in WT, KO, and tKO hPSCs. CDH1 and PXN distribution within colonies is changed in KO and tKO compared to WT, and in KO compared to tKO. DAPI marks the nuclei (blue). The zoomed inset highlights a selected area from the merged image. Scale bar = 50 μm. E High-resolution SIM microscopy images (3D projections) of CDH1 (upper panel) and PXN (bottom panel) immunofluorescence staining in WT, KO, and tKO hPSC colonies. F Representative images of CDH1 (green) show disrupted colony integrity in tKO. N = 3 biological replicates. Scale bar = 100 μm. G High-resolution microscopy images of F-ACTIN (green) immunofluorescence staining show different actin cytoskeleton organization at the colony edges of WT and tKO hPSC. Scale bar = 20 nm. H Western blotting analysis of phospho-AKT (p-AKT), total AKT protein level in WT, KO, and tKO hPSCs. An antibody against GAPDH was used as a loading control. N = 3 biological replicates. I Representative bright-field images of WT and KO hPSCs (cells marked by a yellow mask) after 24 h treatment with PI3K/AKT pathway inhibitors (PI-103, Torin2) or an activator (insulin). Untreated and DMSO-treated WT and KO hPSCs served as controls. Scale bar = 400 μm. J Quantification of crystal violet staining shows a dose-dependent decrease in the attachment of KO (green) after PI3K/AKT pathway inhibition by PI-103, compared to untreated (UT) and control (DMSO) KO hPSCs. N = 4 biological replicates. KO: UT vs. 5 nM, p = 0.0003, UT vs. 15 nM and UT vs. 30 nM, p < 0.0001; 30 nM KO vs. UT WT, p = 0.0422. K Quantification of crystal violet staining shows a dose-dependent decrease in cell attachment following Torin2 treatment, compared to both untreated (UT) and DMSO-treated KO hPSCs. Treatment with 15 nM Torin2 resulted in a proportion of attached KO cells similar to untreated WT (yellow) hPSCs. Treatment with 200 nM Torin2 resulted in decreased attachment of KO compared to WT hPSCs. N = 4 biological replicates. KO: UT vs. 2 nM, p = 0.0003, UT vs. 15 nM, and UT vs. 200 nM, p < 0.0001; 15 nM KO vs. UT WT, p = ns. L Quantification of crystal violet staining shows a dose-dependent increase of WT (yellow) attachment after insulin treatment, compared to untreated (UT) WT hPSCs. The 3 µg/ml insulin treatment resulted in a similar number of attached WT cells as in untreated (UT) KO hPSCs (WT: UT vs. 3 µg/ml, p < 0.0001; KO: UT vs. 3 µg/ml, p = ns). The fraction of KO (green) hPSCs did not change following insulin treatment compared to UT KO hPSCs (all doses, p = ns). No difference in the number of attached KO hPSCs was observed regardless of the insulin dose, indicating that ETV1 deletion may cause saturation of PI3K/AKT pathway activity. N = 4 biological replicates. M Representative images of immunofluorescence of CDH1 (green) and PXN (red) proteins in Torin2 or insulin-treated WT and KO hPSCs. DAPI marks the nuclei in blue. Scale bar = 200 μm. N Quantification of fluorescence intensity (CDH1 + /DAPI) from immunofluorescence images of untreated (UT), DMSO-, Torin2- (TORIN2), and insulin-treated (INS) WT (yellow) and KO (green) hPSCs. N = 3 biological replicates. WT: UT vs. DMSO, p = ns, UT vs. TORIN2, p = 0.0016, UT vs. INS, p = 0.0447; KO: UT vs. DMSO and UT vs. INS, p = ns, UT vs. TORIN2, p = 0.0001. N Quantification of fluorescence intensity (PXN + /DAPI) from immunofluorescence images of untreated (UT), DMSO-, Torin2- (TORIN2) or insulin-treated (INS) WT (yellow) and KO (green) hPSCs. N = 3 biological replicates. WT: UT vs. DMSO, p = ns, UT vs. TORIN2 and UT vs. INS, p < 0.0001; KO: UT vs. DMSO and UT vs. INS, p = ns, UT vs. TORIN2, p < 0.0001. For plots ( J – L, N , and O ), a one-way ANOVA for multiple comparisons was used to determine the p -values shown on the graph. The data are presented as means ± SDs.

Article Snippet: The supernatant was immunoprecipitated overnight at 4 °C with either anti-ETV1 antibody (20 μg; Thermo Fisher Scientific, cat. number: PA5-67447) or control IgG antibody (1 μg; Thermo Fisher Scientific, cat. number: 02-6102) using magnetic protein A/G beads.

Techniques: Immunofluorescence, Staining, Western Blot, Expressing, Control, Microscopy, Inhibition, Cell Attachment Assay, Activity Assay, Fluorescence

Journal: Nature Communications

Article Title: ETVs dictate hPSC differentiation by tuning biophysical properties

doi: 10.1038/s41467-025-56591-6

Figure Lengend Snippet: A Scheme illustrating the micropatterning approach to in vitro generation of the organized embryo-like structures (gastruloids). hPSCs (light blue) were plated on laminin 521-coated micropatterns and exposed for 46 h to BMP4 to induce self-organization and formation of gastruloids, containing extraembryonic-like cells (red), ectodermal cells (green) and the mesodermal and endodermal cells (dark blue). B Representative immunofluorescence staining of ISL1 (red), SOX2 (green), and BRA (blue) in gastruloids generated from ESI (hPSC control cell line), HUES8 (hPSC control cell line), iCas9 (WT hPSCs, parental control), KO and tKO hPSCs. Note, the disrupted germ layers organization in KO hPSCs (SOX2+ and BRA+), the overgrowth of extraembryonic-like cells (ISL1+), and the absence of SOX2+ cells in tKO, compared to control hPSC lines (ESI, HUES8 and iCas9). Scale bars = 200 µm. C Distribution from the center to the gastruloid edge of ISL1, SOX2, and BRA positive signals based on immunofluorescence staining on ESI (blue), HUES8 (red), iCas9 (green), tKO (black), and KO (pink) gastruloids. Data are presented as the mean ± SEM. N = 3 biological replicates. D WT, KO, and tKO differentiation to definitive endoderm using a standard protocol with full doses of Activin A and CHIR99021 (a WNT activator). N = 3 biological replicates. KO vs. WT, p = ns, tKO vs. WT, p = 0.0232, tKO vs. KO, p = 0.0002. E WT, KO, and tKO differentiation to definitive endoderm using a suboptimal protocol with decreased concertation of Activin A, with or without CHIR99021. N = 3 biological replicates. KO vs. WT and tKO vs. KO, p < 0.0001, tKO vs. WT, p = ns. F Scheme illustrating hPSC spontaneous differentiation into embryoid bodies (EBs). G Gene expression changes in KO EBs reveal altered differentiation potential. Dot plot shows log2FC in gene expression (qPCR) between KO and WT EBs at days 8 and 10 of spontaneous differentiation. The increased expression of endodermal and mesodermal markers and decreased expression of ectodermal markers were observed in KO at both timepoints. N = 3 biological replicates. The p -values were determined by two-sided student’s t -test. H Representative immunofluorescence images of human fetal pancreas at weeks 10.6 and 13 of embryogenesis stained with PDX1 (red), CHGA (gray), and ETV1 (green). N = 3 tissue samples. Scale bar = 100 µm. I Representative immunofluorescence staining of ETV1 (green) in WT hPSC-derived definitive endoderm (DE, upper panel), and pancreatic progenitors (PP, bottom panel). SOX17 (red) marks DE, and PDX1 (green) and NKX6-1 (red) mark PPs. N = 3 biological replicates. Scale bar = 200 µm. J Scheme illustrating hPSC differentiation into pancreatic progenitors (PP). During differentiation, hPSCs pass through the following stages: definitive endoderm (DE) marked by FOXA2 and SOX17 expression, PP marked by PDX1 and NKX6-1 expression. At the PP stage (day 12) single-cell RNA sequencing and ATAC sequencing were performed on WT and KO samples. K Representative immunofluorescence staining of PDX1 (green), NKX6-1 (red), and CHGA (gray) proteins in WT and KO PPs. N = 3 biological replicates. Scale bar = 200 µm. L Representative flow cytometry analysis showing the absence of NKX6-1+KO PPs and decreased PDX1 protein levels in KO compared to WT PPs. Two distinct PDX1+ populations are observed in KO cells: low PDX1+ (~50% of cells) and high PDX1+ (~50% of cells). In contrast, WT PPs exhibit predominantly high PDX1 expression. For plots ( D , E ), a one-way ANOVA with Turkey’s correction for multiple comparisons was used to determine the p -values shown on the graph. The data are presented as means ± SDs.

Article Snippet: The supernatant was immunoprecipitated overnight at 4 °C with either anti-ETV1 antibody (20 μg; Thermo Fisher Scientific, cat. number: PA5-67447) or control IgG antibody (1 μg; Thermo Fisher Scientific, cat. number: 02-6102) using magnetic protein A/G beads.

Techniques: In Vitro, Immunofluorescence, Staining, Generated, Control, Gene Expression, Expressing, Derivative Assay, RNA Sequencing, Sequencing, Flow Cytometry