Fig. 3: Effects of PRMT1-KO on the differentiation of pancreatic EPs into ECs.

a Schedule for the dox treatment of PEs differentiated from P-iKO hESCs (yellow line). Dox was administered for 3 d to PRMT1-iKO hESCs that had progressed to D3 of the PE stage of pancreatic lineage development. See also Supplementary Fig. 2d–f. b Expression of PRMT1 and NGN3 in P-iKO EPs treated with dox. Relative PRMT1 and NGN3 protein expression levels were normalized to GAPDH levels. The H4R3me2a expression levels were normalized to H3 levels. The data are presented as the mean ± SEM. *p < 0.05, ***p < 0.001 (n = 3–5). See also Supplementary Fig. 3a. c Immunostaining of NGN3 in P-iKO EPs treated with dox (left). NGN3-positive cells were counted using ImageJ (right). P-KO EPs accumulate NGN3. The data are presented as the mean ± SEM. ***p < 0.001. Dox(−), n = 27; dox(+), n = 20. Scale bars, 50 μm. See also Supplementary Figs. 3–5. d Expression of PRMT1 and NGN3 in P-iKO EPs treated with dox. Western blot analysis showed reduced PRMT1 and enhanced NGN3 levels in P-KO EPD4 and ECD1 cells. PRMT1 and NGN3 protein expression levels were normalized to those of GAPDH. Dox(+) and dox(-) indicate treated and untreated cells, respectively. The data are presented as the mean ± SEM. *p < 0.05, **p < 0.01 (n = 4). e Relative mRNA expression of EP-associated genes in P-KO EPs. The transcription of NGN3 target genes was significantly downregulated in P-KO EPs. The data are presented as the mean ± SEM. *p < 0.05, ***p < 0.001 (n = 4). See also Supplementary Fig. 3d–g. f Relative mRNA expression of EC-associated genes in P-KO ECs. Transcripts of NGN3 target genes and EC-associated genes were significantly decreased in P-KO ECs. The data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 (n = 3). See also Supplementary Fig. 3h, i. g Relative mRNA expression of endocrine hormone genes in P-KO ECs. INS and SST genes were transcriptionally downregulated in P-KO ECs. The data are presented as the mean ± SEM. ***p < 0.001 (n = 4). INS, insulin; PPY, pancreatic polypeptide; SST, somatostatin. h Immunostaining of endocrine hormones in P-KO ECs. GCG-positive cells were not observed. Scale bars, 50 μm. i Cytoplasmic and nuclear fractionation of endogenous NGN3 in P-KO EPs. P-KO EPs accumulated NGN3 in the cytoplasm. The data are presented as the mean ± SEM. WCL, whole-cell lysate; Cyt, cytoplasm; Nuc, nucleus; n.s., not significant. *p < 0.05 (n = 3). See also Supplementary Figs. 4e, 5.