Fig 2: The expression of proteins required for exocrine development and function are significantly altered in the absence of hypusine biosynthesis. A, Western blot analysis of E18.5 pancreata harvested from Ptf1a‐cre (control), Dhps ΔPANC, and Eif5a ΔPANC mice. DHPS, total eIF5A (eIF5ATOT), hypusinated eIF5A (eIF5AHYP), CPA, Elastase, and Pdx1 expression was measured. B, Densitometry values were normalized to total protein input as detected by Revert. Relative protein expression levels are shown in the bar graphs. Data are presented as mean ± SEM, n = 3/group, *P < .05, **P < .005, ****P < .0001. C,D, Volcano plots displaying proteomic data from the Dhps ΔPANC or Eif5a ΔPANC mutants compared with Ptf1a‐cre controls. Each dot represents a protein; those in the shaded areas were identified as significantly up‐ (red) and down‐ (green) regulated in the mutants with a minimum fold change of 1.5 and P < .05. E, Proteins identified as differentially expressed in both the Dhps ΔPANC and Eif5a ΔPANC compared with the Ptf1a‐cre control

Fig 3: Differentiation-specific hyper-DMRs show reduced chromatin activity at pioneer TF-binding sites.a Volcano plot of WGBS data illustrating differentially methylated CpGs (DMCs) identified in TKO_PP compared with WT_PP. Red and blue represent increased and decreased 5mC in TKO_PP cells, respectively (credible methylation difference >0.2). b Heatmap illustrating methylation difference between TKO_PP and WT_PP at centers of annotated genomic features (±5 kb) for chromatin accessibility (ATAC), hydroxylation (5hmC), TF binding (FOXA2, GATA4, and GATA6), bivalent promoters, poised enhancers, and active enhancers. Average 5mC signals of every 100-bp bin were calculated. c Classification of TKO hyper-DMRs based on 5mC levels in hESCs (green), WT_PP cells (blue), and TKO_PP cells (red). d Average density plots and heatmaps of FOXA2, GATA4, GATA6, PDX1, and HNF6 signals at differentiation-specific hyper-DMRs or non-differentiation hyper-DMRs in pancreatic progenitors. The statistical significance was calculated using the student’s unpaired two-tailed t-test without multiple test correction. e Average density plots and heatmaps of ATAC-seq reads at differentiation-specific hyper-DMRs or non-differentiation hyper-DMRs in PP for WT (green) or TKO (orange) cells. f Enrichment profile of methylation ratio (5mC/C) at proximal (≤1 kb from TSS) and distal (>1 kb from TSS) decreased accessible regions in PP for WT (green) and TKO (orange) cells. g Genome-browser view of the PDX1/PDX1-AS1 locus. A specific TKO hyper-DMR showing decreased 5hmC, ATAC-seq, and H3K27ac signals is highlighted in pink.

Fig 4: Expression of FGFR2 in hFP. (A): Cells co‐expressing PDX1 (red), SOX9 (light blue), and FGFR2 (green) are present at the tips of the branching epithelium (arrowheads). (B): Cells co‐expressing SOX9 (red), PTF1A (light blue), and FGFR2 (green) are found at the tips of the pancreatic epithelium (arrowheads). Cells co‐expressing SOX9 and PTF1A, but not expressing FGFR2, are also present at the termini of the branches (asterisks). be = branching epithelium. Scale bar: 25 μm, hFP: 13.5WGA. (C): Sorting strategy for the isolation of subpopulations. Cells were gated based on the expression of SOX9 and PTF1A, detected through RNA probes, and gated for the expression of FGFR2. SOX9+/PTF1A+/FGFR2+ (P8) cells represent a small fraction (0.1%) of the total hFP cell population compared to SOX9+/PTF1A+/FGFR2− (P7) cells, which constitute on average 15% of total hFP. The negative fraction of cells (SOX9−/PTF1A−, Q3) was also gated on FGFR2, confirming that cells expressing FGFR2 (SOX9−/PTF1A−/FGFR2+, P6) represent only 0.1%, whereas the majority of SOX9−/PTF1A− cells are also FGFR2− (SOX9−/PTF1A−/FGFR2−, P5). (D): Distribution of SOX9+/PTF1A+/FGFR2− and SOX9+/PTF1A+/FGFR2+ subpopulations at 14 and 17WGA. (E): Given the very low expression of FGFR2, specificity of the FGFR2 antibody was tested by using a mouse IgG1 isotype control on total pancreatic digestion. In order are: an unstained control, a sample stained with FGFR2 antibody (staining 1.7% of the cells), and a sample stained with a mouse IgG1 isotype (showing 0.1% of nonspecific signal).

Fig 5: MAFB is expressed in both human alpha and beta cells.(A) Different antibodies were tested for their specificity toward mouse MafB by triple immunofluorescent (IF) staining of mouse pancreatic sections. Representative images of triple IF staining with anti-glucagon, insulin, and hMAFA, mMafB1, hMAFB2 and hMAFB3 antibodies. Note that hMAFB3 did not show an alpha cell specific pattern as did mMafB1 and hMAFB2 antibodies. (B) MAFB expression was detected in both human pancreatic alpha and beta cells. Representative images of triple IF-staining showing MAFB expression in human islets from healthy individuals. The percentages of positive cells for MAFB in glucagon+ (green) and insulin+ (red) cells were 67.2±7.6% MAFB+ alpha cells and 53.6±10.5% MAFB+ beta cells. Results are the averaged expression ± S.E.M of counting results from n = 4 control individuals (463 GLU+ cells and 797 INS+ cells were counted in total). Scale bar = 25 µM. (C) MAFB expression in insulin+ PDX1+ cells. The percentage of double insulin+ PDX1+ cells positive for MAFB was 30.2±5.8% (858 INS+ PDX1+ counted cells in total). The above percentages of expression are the averaged counting results ±S.E.M from n = 3 control individuals. Scale bar = 25 µM.