Fig. 5: Demethylation activity of ALKBH5 is required for human pancreatic differentiation.

a A schematic representation of the rescue experiment. WT + GFP^N, A5-KO + GFP^N, A5-KO + A5^WT, and A5-KO + A5^Mut hPSC lines were used. b RT-qPCR analysis of the expression of ALKBH5 (n = 3 biological replicates). c Western blotting analysis of ALKBH5 protein levels in WT + GFP^N, A5-KO + GFP^N, A5-KO + A5^WT, and A5-KO + A5^Mut hPSC lines. ACTIN was used as a loading control. Images are representative of three independent replicates. d The quantification of m⁶A abundance in WT + GFP^N, A5-KO + GFP^N, A5-KO + A5^WT, and A5-KO + A5^Mut hPSC lines by UHPLC-QQQ-MS/MS (n = 4 biological replicates). e Immunofluorescent staining of PDX1, NKX6.1, and nuclei. Images are representative of three independent replicates. Scale bar, 100 μm. f Representative flow cytometry plots and the percentage of PDX1⁺NKX6.1⁺ cells in WT + GFP^N, A5-KO + GFP^N, A5-KO + A5^WT, and A5-KO + A5^Mut PP populations (n = 3 biological replicates). g RT-qPCR analysis of PDX1, NKX6.1, SOX9, and HNF6 expression (n = 3 biological replicates). All data are presented as mean ± s.d. Statistical significance calculated using two-tailed Student’s t-test, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Source data are provided as a Source Data file.