Fig. 2: GPX2 deficiency results in altered differentiation of the posterior foregut endoderm.

A Design and validation of GPX2 knockout (KO) in hPSCs. Three sgRNAs (arrows) were used to delete exons 1 and 2 (orange blocks) of the GPX2 gene. A representative gel electrophoresis image confirms the deletion at the DNA level. Western blot analysis validates the absence of GPX2 protein in KO (clonal line KO1) pancreatic progenitors (PP) and endocrine progenitors (EP). GAPDH was used as a loading control. B Changes in mitochondrial and cytoplasmic oxidative stress levels in KO (clonal line KO1) pancreatic endoderm (PEN) compared to WT PEN, assessed by flow cytometry. Mean and 95% confidence intervals (CIs) are indicated. N = 3 independent experiments. An unpaired, two-sided t-test was used to determine statistical significance. C Representative IF staining for SOX17 (red) and FOXA2 (green) in WT and KO definitive endoderm (DE). DAPI marks nuclei (blue). Scale bar = 100 µm. Quantification of the mean fluorescence intensity (MFI) of SOX17 relative to DAPI for WT (orange) and KO (clonal line KO1, blue) spheroids is shown as a dot plot. Each dot represents one image, with mean and 95% confidence intervals (CIs) indicated. N = 3 independent experiments. An unpaired, two-sided t-test was used to determine statistical significance. D Representative IF staining for SOX17 (green) and HHEX (red), or CDX2 (green) and HNF4A (red) proteins in WT and KO (clonal line KO1) posterior foregut (PFG). DAPI marks nuclei (blue). Scale bar = 100 µm. Quantification of marker mean fluorescence intensity (MFI) relative to DAPI for WT (orange) and KO (blue) spheroids is shown as a dot plot. Each dot represents one image, with mean and 95% confidence intervals (CIs) indicated. N = 3 independent experiments. An unpaired, two-sided t-test was used to determine statistical significance. p-value for each WT vs. KO comparison were: SOX17: p = 0.0034, HHEX: p < 0.0001, CDX2: p = 0.0017, HNF4A p = 0.0014. E Representative IF staining for PDX1 (red), SOX9 (green), or CDX2 (red) and HHEX (red) proteins in WT and KO (clonal line KO1) pancreatic endoderm (PEN). DAPI marks nuclei (blue). Scale bar = 100 µm. Quantification of the marker mean fluorescence intensity (MFI) relative to DAPI for WT (orange) and KO (blue) spheroids is shown as a dot plot. Each dot represents one image, with mean and 95% confidence intervals (CIs) indicated. N = 3 independent experiments. An unpaired, two-sided t-test was used to determine statistical significance. p-value for each WT vs. KO comparison were: PDX1: p < 0.0001, SOX9: p < 0.0001, CDX2: p = 0.0083, HHEX: p < 0.0001. F Zoom inserts show SOX17 (green) and HNF4A (red) co-expression in WT and KO posterior foregut (PFG), and HNF4A (red) and FGB (green) in KO (clonal line KO1) PFG. Cells co-expressing HNF4A and FGB are indicated by yellow arrows. DAPI staining marks nuclei (blue). Scale bar = 100 µm. Due to low FGB presence in WT PFG cells, as shown by FC analysis in (H), co-staining for HNF4A and FGB is shown only in KO cells. N = 3 biological repeats. G Zoom inserts show HNF4A (green), PDX1 (red), and FGB (gray) expression in KO (clonal line KO1) pancreatic endoderm (PEN). The merged image includes DAPI-stained nuclei (blue). The yellow arrow marks a cell co-expressing HNF4A and FGB but not PDX1. Scale bar = 50 µm. Due to low FGB presence in WT PFG cells, as shown by FC analysis in (H), co-staining for HNF4A, PDX1, and FGB is shown only in KO cells. N = 3 biological repeats. H Representative flow cytometry analysis shows the expression levels of HNF4A (APC-H) and FGB (FITC-H) proteins in WT and KO (clonal line KO2) at PFG and PEN stages. In KO, a higher percentage of cells co-express HNF4A and FGB at both stages compared to the WT counterpart. Source data are provided as a Source data file.