A. Expression of lacZ knocked into the null allele of Ifrd1+/− mice after TAM showing induction of expression in basal chief cells early in paligenosis. Scale bar, 50μm.
B. RNAScope™ shows Ifrd1 mRNA in chief cells after TAM. Scale bar; inset highlights two red puncta from boxed area; arrows indicate other puncta, Scale bar 50μm.
C. Western blot for DDIT4 and IFRD1 and mTORC1 proxy pS6 240/244, tubulin as loading control.
D. qRT-PCR for Ifrd1 and Ddit4. Each datapoint represents mean±SEM from n=3 mice cohorts.
E. Proliferation measured by BrdU 72h after TAM. Scale bar, 50μm.
F. Data as in panel (E) quantified, focusing on the basal 100 μm gastric of Ifrd1+/+ (n=6) and Ifrd1–/– (n=6) mice gastric unit. Datapoint: Mean±SEM of ≥40 glands quantified per mouse with significance estimated by unpaired, two-tailed t-test.
G. As per panel (F) but for SOX9+ progenitor cells of Ifrd1+/+ (n=3) and Ifrd1–/–(n=3) mice.
H. Organoids from stomach body at various days after isolation. Scale bar, 100μm. Initial outgrowth of wildtype organoids occurs throughout the gastric unit, whereas the bases (arrow) of Ifrd1−/− mice do not expand, slowing overall organoid growth
I. Data as in panel (H), quantified for organoid diameter after 5 day in culture of Ifrd1+/+ (n=3) and Ifrd1–/–(n=3) mice. Datapoint: Mean±SEM of ≥100 gastroids per mouse with significance estimated by unpaired, two-tailed t-test.
J. BrdU (green) identifying proliferating acinar cells 5 days post cerulein-injury-induced paligenosis (acinar cells labeled by digestive enzyme amylase, red). Yellow arrows=representative paligenotic cells; white arrows=non-epithelial cells. Scale bar, 50μm
K. Paligenotic (ie amylase-positive) proliferative (ie BrdU+) cells of Ifrd1+/+ (n=3) and Ifrd1–/– (n=3) mice from panel (J) are quantified. Datapoint: Mean±SEM of ≥10 random fields per mouse with significance estimated by unpaired, two-tailed Student’s t-test.
L. As per panel (K) but with quantification of SOX9+ progenitor cells of Ifrd1+/+ (n=3) and Ifrd1–/–(n=3) mice.