Fig. 4: Prolonged retinoic acid exposure of early pancreatic progenitor cells phenocopies LSD1 inhibition.

a Enriched transcription factor (TF) binding motifs with associated p-values for G1 enhancers compared to G2 and G3 enhancers. Fisher’s exact test, 2 sided, corrected for multiple comparisons. b Enrichment for RXR peaks (±1 kb) among G1 enhancers versus random genomic regions. P = 0, permutation test. c Experimental plan to extend retinoic acid (RA) exposure through PP1 to PP2 (RA^extended) during hESC differentiation to the endocrine cell stage (EN). d Immunofluorescent staining for insulin (INS), glucagon (GCG) and somatostatin (SST) in control EN cells compared to EN cells with extended RA treatment (RA^extended) (representative images, n = 3 independent differentiations). Scale bar, 50 µm. e Flow cytometry analysis at EN stage for NKX6.1, PDX1 and INS comparing control and RA^extended cultures. Isotype control for each antibody is shown in red and target protein staining in green. Percentage of cells expressing each protein is indicated (representative experiment, n = 2 independent differentiations). f Volcano plot of differentially expressed genes at PP2 in RA^extended cultures. Differential expression calculated with DESeq2 and genes with ≥1.5-fold change up or down. Adjusted p-values < 0.05 were considered differentially expressed. 96 genes were downregulated and 69 were upregulated in RA^extended cultures. Black dots indicate genes not significantly changed (p-value > 0.05), grey dots genes significantly changed (p-value < 0.05) but less than 1.5-fold compared to control, red and green dots genes significantly up- and downregulated (p-value < 0.05 and ≥ 1.5-fold change), respectively. Yellow dots highlight genes also upregulated after LSD1 inhibition from PP1 to PP2 (TCP, LSD1i^early) (n = 2 replicates from independent differentiations). g Enrichment analysis of genes associated with LSD1-bound enhancers and upregulated by LSD1i^early among those upregulated by RA^extended. Dashed line indicates p-value = 0.05, Fisher’s exact test, 2 sided. h mRNA levels of select genes significantly upregulated in both LSD1i^early and RA^extended PP2 cells. Levels at PP1 stage are also displayed. Data shown as mean FPKM ± S.E.M. (n = 2 replicates from independent differentiations; source data are provided as a Source Data file). GT, primitive gut tube; PP1, early pancreatic progenitors; PP2, late pancreatic progenitors.