Fig. 2

Biallelic mutation of GLIS3 affects pancreatic differentiation and the generation of endocrine cells. a qRT-PCR analysis of pancreatic markers in the isogenic WT and GLIS3^−/− PP2 cells. (PDX1, NEUROD1, NKX6.1 n = 4, MAFA n = 12). b Heatmap representing the relative expression levels of endocrine genes in WT and GLIS3^−/− PP2 cells (n = 3). c GSEA analysis showing the decrease of endocrine pancreas-related genes in GLIS3^−/− PP2 cells. Gene ontology (GO) analysis (d) and KEGG pathway analysis (e) of genes significantly downregulated (P < 0.01) in GLIS3^−/− PP2 cells. f Flow cytometry analysis of WT and GLIS3^−/− cells at D30_L. g Quantification of the percentage of INS-GFP⁺ cells of WT (n = 6) and GLIS3^−/− (n = 9) cells at D30_L. The percentage of INS-GFP⁺ cells was quantified based on flow cytometry analysis of GFP⁺ cells. h Quantification of the percentage of INS⁺ cells of WT (n = 11) and GLIS3^−/− (n = 14) cells at D30_L. The percentage was quantified based on intracellular flow cytometry analysis of INS⁺ cells. i Total percentage of endocrine cells in isogenic WT (n = 10) and GLIS3^−/− (n = 12) cells at D30_L. The percentage of endocrine cells is calculated as the sum of the percentages of INS⁺, GCG⁺, SST⁺, and GHRL⁺ cells. j Plot representing the ratios of different endocrine subtypes in WT and GLIS3^−/− cells at D30_L. k Immunocytochemistry analysis of pancreatic endocrine marker expression in WT and GLIS3^−/− cells at D30_L. Scale bar = 100 μm. l, m Histogram showing fluorescence intensity (l) and quantification of median fluorescence values (m) of INS staining of WT and GLIS3^−/− PP2-β cells (n = 4). n Insulin content of the purified INS-GFP⁺ WT and GLIS3^−/− PP2-β cells (n = 4). Data are normalized to the WT mean value. P values by unpaired two-tailed t-test were *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. The center value is “mean”. Error bar is SEM