Fig 2: Cellular alterations in CA-PKA mutant stomach.A–D, Stomach from 6-week- and 12-month-old control and CA-PKA mice co-stained with lectins UEA1 (red) and GSII (green). In control (A, C), UEA1 in pit cells (arrows) and GSII signal in mucous neck cells (arrowheads) are shown. In mutant (B, D) UEA1/GSII double-positive cells (arrowheads) and cystic glands (dotted lines) containing both UEA1+ and GSII+ cells (arrows) are shown. E–F, Stomach from 12-month-old control (E) and CA-PKA (F) mice co-stained with MUC5B (red) and GSII-lectin (green). In control, MUC5B (arrows) and GSII (arrowheads) stained distinct cells. In the CA-PKA corpus, increased MUC5B/GSII double-positive cells (arrowheads) and remaining MUC5B+/GSII- cells (arrows) are shown. G, Representative immunoblots and quantification graphs (*P < .05) showing gastrokine1 (n = 3), TFF2 (n = 4), and MUC5B (n = 4) relative levels in corpus extracts of control and CA-PKA mice. H–K, Stomach from control and CA-PKA mutant co-stained with MIST1 (red) and DBA-lectin (green). In controls (H, J), MIST1+ chief cells (arrowheads) and DBA+ parietal cells (arrows) are shown. In 6-week-old mutant (I) DBA+ (arrows) and MIST1+ cells (arrowheads) were fewer. In 12-month-old mutant (K), glands containing severely reduced (arrows) or no DBA+ cells (arrowheads) are indicated. L–M, Stomach sections from control and mutant co-stained with parietal cell marker, ATP4A (red, arrows) and GSII-lectin (green, arrowheads). In control (L) ATP4A+ (arrows) and GSII+ (arrowheads) cells are indicated. Mutant glands (M) containing severely reduced (arrowheads) or no ATP4A+ cells (arrows) are indicated. N, Representative immunoblots and quantification graphs (*P < .05) showing ATP4A (n = 4) and MIST1 (n = 3) relative levels in mutant corpus vs control. Scale bars = 50µm. Blue (DAPI).

Fig 3: Lineage tracing and scRNA-seq by Gif+ chief cells identify injury-responsive chief cells(A) Diagram of the Gif-Cre-nTnG allele used in this study.(B) Experimental scheme of the L635 treatment study in Gif-Cre-nTnG mice. Dox was administered for 1 week and off for 2 weeks to label chief cells with GFP. The mice were then left untreated or treated with 3 doses of L635.(C and D) Paraffin sections of Gif-Cre-nTnG mouse stomachs untreated or treated with 3 doses of L635 were immunolabeled with antibodies against (C) GFP (green), Mist1 (chief cells, red), GSII-lectin (neck cells, blue), and P120 (epithelial cell membrane, white) or (D) GFP (green), p57 (red), and Ki67 (proliferating cells, white). GFP+Mist1+ cells indicate chief cells and GFP+GSII+ cells indicate injury-responsive chief cells. Arrowheads indicate GFP+p57+ double-positive cells (yellow) and GFP+Ki67+p57-proliferating injury-responsive chief cells (red), respectively. Nuclei were counterstained with hoechst (blue) in (D). Yellow boxes indicate enlarged areas. Orange-dotted boxes indicate enlarged areas shown in Figure S3C. Scale bars, 100 µm.(E) UMAP plots of scRNA-seq from Gif lineage cells (GFP+TdTom-) of Gif-Cre-nTnG mice in both conditions (Total, left), uninjured (Control, middle), and 2 days after DMP-777-induced corpus injury (Injury, right). Gifhigh and Giflow populations are marked by brown-dotted lines.(F) UMAP plots for control (light blue) and injury (red) cells.(G) UMAP plots of Gif expression in control (left) and injury (right).(H) Dot plot for expression of marker genes of each cell type in Gifhigh and Giflow cell populations.(I) Dot plot for expression of marker genes of proliferation or cell cycle in Gifhigh and Giflow cell populations.(J) Projection of Gifhigh and Giflow cells on the UMAP plot for Pgc+ scRNA-seq. The cells in control and injury are denoted as light blue and red, respectively. Brown-dotted circles indicate the injury-responsive chief cell population.(K) UMAP plots of p57 and Ki67 expressions in control (left) and injury (right).See also Figure S3.