Figure 2. Expanded Neurog3 lineage and β-like cells in gut of Neurog3 FoxO1-KO mice.

(A) FACS of isolated Tomato⁺ cells from either Neurog3Cre⁺FoxO1^fl/fl; ROSA^tdTomato (NFKO) or Neurog3Cre⁺; ROSA^tdTomato (WT) gut epithelial cells. Red gate indicates sorting window for Neurog3-derived Tomato⁺ cells. (B) Tomato⁺ cell frequency assessed by FACS in NFKO and Neurog3Cre (WT) mice (NFKO, n = 23; WT, n = 16 mice). Data are represented as mean ± SEM. Two-tailed t test. (C) Representative IHC images of 2 types of Neurog3-derived β-like cells from NFKO mice: Paneth pattern (upper panel) and EEC pattern (lower panel). Scale bars: 40 μm. (D) FACS plot of CD24-based sorting strategy of dissociated Tomato⁺ single cells from NFKO small intestinal epithelial cells. SSC, side scatter. (E) Ins1 and Ins2 mRNA in sorted CD24⁺Tomato⁺, CD24^negTomato⁺, and Tomato^neg populations (n = 4 mice, Mann-Whitney rank-sum test). (F) Representative IHC of insulin, CD24, and Tomato. Paneth (upper panels) and EEC pattern (lower panels) of CD24 staining in insulin⁺ cells (green and red channel double colocalization is shown in yellow; green, red, and magenta triple colocalization is shown in white). (G and H) Enriched hallmark gene sets in CD24^negTomato⁺ versus Tomato^neg population predicted by the GSEA.