Fig. 6. The initial suppression of mTORC1 and autophagic/lysosomal recycling of cell architecture in paligenosis Stage 1 depends on DDIT4.

A. DDIT4 (green) is confined to gastric chief cells, increasing in Stage 1 (4–24h) then lost later. GSII (red, a progenitor marker) labels Stage 2–3 paligenotic chief cells at base of gland and normal progenitor cells higher in the gland. As paligenotic cells become GSII+, they lose DDIT4, indicating DDIT4 is in homeostatic chief cells, increased in Stage 1, and lost thereafter. Blue=nuclei. Scale bar, 30 μm; boxed area insert highlighting base, 15 μm.

B. Anti-pS6 240/244 showing that at mid-Stage 1 (12h), Ddit4^−/− paligenotic cells fail to extinguish mTORC1. Scale bar, 30 μm; boxed area insert highlighting base, 15 μm.

C. Representative western blot for pS6 240/244 showing decreased mTORC1 in Stage 1 (12h) only in Ddit4^+/+ mice. Note pS6 is also in non-paligenotic cells, so a blot of whole stomach body registers only partial decrease. This blot is one of those quantified in panel (D).

D. Western blots quantified from n=3 independent mouse cohorts represented as mean±SEM, significance estimated by unpaired, two-tailed Student’s t-test at the 12h timepoint.

E. H&E staining of stomachs in Ddit4^+/+ and Ddit4^−/− mice at 72h after tamoxifen injury (TAM 72h). Red arrows show representative glands with dropout (ie the cells at the base of units are lost) in Ddit4^+/+ control. Note that cells are mostly cuboidal/columnar. There is no dropout apparent in Ddit4^−/− mice; black arrows show representative bases with most chief cells preserving their cytoplasm 72h after tamoxifen injury. Scale bar, 30 μm; boxed area insert, 15 μm.

F. Western blot of GIF and β-tubulin control expression from whole corpus protein extracts of Ddit4^+/+ and Ddit4^−/− mice at various time points after tamoxifen injury.

G. GIF (red, chief cells), GSII (green, progenitor or paligenotic cells), BrdU (white), DAPI (blue). Note paligenotic cells Ddit4^−/− mice are both more proliferative (BrdU+) and have more GIF⁺ granules, indicating failed Stage 1 downscaling. Scale bar, 30 μm; boxed area insert, 15 μm.

H. Proliferating (BrdU+) cells of Ddit4^+/+ (n=3) and Ddit4^−/− (n=3) mice quantified by cell type in Stage 3, proliferative paligenosis (72h TAM). GIF-negative cells lack chief cell markers so are non-paligenotic: ie derived from GSII+ progenitors from neck (green) or progenitors even higher in the unit (gray); GIF+GSII− cells aberrantly retain chief cell features without inducing progenitor markers, indicating failed Stage 1. Ddit4^−/− mice have significantly increased paligenotic (GIF+GSII+) and aberrant paligenotic cells (GIF+) but no difference in non-paligenotic proliferating cells. Datapoint: mean±SEM across ≥40 fields, significance estimated between paired cell types by unpaired, two-tailed t-test.

I. Total BrdU⁺ (proliferating) cells from Ddit4^+/+ (TAM 24h, n=3; TAM 48h, n=3; TAM 72h, n=8) and Ddit4^−/− (TAM 24h, n=3; TAM 48h, n=3; TAM 72h, n=8) mice per unit were quantified, Datapoint: Mean±SEM of ≥40 glands quantified per mouse, significance estimated by unpaired, two-tailed t-test at each timepoint.

J. Organoids at day 6 after culturing from Ddit4^−/− and Ddit4^+/+ mice. Scale bar, 100μm.

K. Data as in panel (J), quantifying organoid diameter from Ddit4^+/+ (n=3) and Ddit4^−/− (n=3) mice. Datapoint: Mean±SEM of ≥100 gastroids per mouse with significance estimated by unpaired, two-tailed t-test.

L. Anti-pS6 240/244 showing normal, homeostatic mTORC1 in Ddit4^+/+ and Ddit4^−/− pancreas, with Ddit4^−/− acinar cells failing to extinguish mTORC1 at cerulein injury day 1 (mid-Stage 1 in pancreas); inset in Ddit4^+/+ highlights a rare pS6+ cell, whereas most cells are positive in the absence of DDIT4. Scale bar, 30 μm; boxed area insert, 15 μm.

M. H&E staining of Ddit4^+/+ and Ddit4^−/− pancreas after 5 days of cerulein treatment (CER D5). Most acinar cells preserve their cytoplasm in Ddit4^−/− mice, consistent with failure of Stage 1 turnover of mature cell architecture. Scale bar, 30 μm; boxed area insert, 15 μm.

N. Anti-BrdU of pancreas 5d after CER injury. Note increased BrdU+ cells in Ddit4^−/− mice and failed downscaling of cell architecture. Scale bar, 30 μm; boxed area insert, 15 μm.

O. BrdU-positive cells from Ddit4^+/+ (n=6) and Ddit4^−/− (n=6) mice as in panel (N) quantified. Datapoint: Mean±SEM of ≥10 random fields quantified per mouse with significance estimated by unpaired, two-tailed Student’s t-test.