Fig. 3: ALKBH5 is essential for pancreatic lineage specification.

a Western blotting analysis of ALKBH5 protein levels in hPSCs, DE, PPs, and hILOs. ACTIN was used as a loading control. Images are representative of three independent replicates. b A schematic representation of the generation of A5-KO hPSCs using CRISPR-Cpf1. c Western blotting analysis of ALKBH5 protein levels in WT and A5-KO hPSCs. Images are representative of three independent replicates. d A schematic representation of the differentiation of WT and A5-KO hPSCs to PP stage. e Immunofluorescent staining of WT and A5-KO PPs for PDX1, NKX6.1, and nuclei (Hoechst). Images are representative of six independent replicates. Scale bar, 100 μm. f Representative flow cytometry plots and the percentage of PDX1 and NKX6.1 double-positive PPs in WT and A5-KO populations (n = 3 biological replicates). g The expressions of PP marker genes PDX1, NKX6.1, HNF6, and SOX9 in WT and A5-KO PPs (n = 3 biological replicates). h Representative western blotting of NKX6.1 protein in WT and A5-KO PPs. ACTIN was used as a loading control. Images are representative of three independent replicates. i Volcano plot of differentially expressed genes in A5-KO PPs versus WT PPs. Red, upregulated genes (n = 965, FC > 1.5, p < 0.05); blue, downregulated genes (n = 811, FC < 0.67, p < 0.05). j Heatmap visualization for the expression of upregulated and downregulated marker genes. k GO analysis of upregulated and downregulated genes, p-values were calculated by one-tailed hypergeometric test. All data are expressed as mean ± s.d. Statistical significance calculated using two-tailed Student’s t-test, * p < 0.05, ** p < 0.01, *** p < 0.001. Source data are provided as a Source Data file.