Fig. 7. mTORC1 inhibition rescues Ddit4^−/− Stage 1 phenotype.

A. Rapamycin blocks pS6 (mTORC1) in Ddit4^−/− and Ddit4^+/+ mice. Scale bar, 30 μm; boxed area highlighting bases of units where paligenosis occurs; inset, 15 μm.

B. Western blot of whole corpus ±rapamycin. Note IFRD1 is less abundant at 72h, so exposure was longer than for Fig. 2c.

C. As in panel (A) but with anti-BrdU. Paligenotic proliferation (BrdU) in Ddit4^−/− stomachs is corrected by rapamycin, such that both genotypes have the usual loss of proliferation upon mTORC1 reactivation. Scale bar, 30 μm; boxed area insert, 15 μm.

D. Data from Ddit4^+/+ (n=5), Ddit4^−/− (n=5), Ddit4^+/++ rapamycin (n=5) and Ddit4^−/−+ rapamycin (n=5) mice as for panel (C) were quantified. Note rapamycin does not significantly affect non-paligenotic proliferation, so only paligenotic proliferation is inhibited. Datapoint: Mean±SEM of ≥40 glands quantified per mouse, significance by one-way ANOVA, Dunnett post-hoc to Ddit4^+/+ control.

E. Immunohistochemistry images of SOX9 staining after 5 days cerulein injury (CER D5) ± rapamycin (RAP).

F. Quantification of immunostaining depicted in panel (E) from Ddit4^+/+ (n=5), Ddit4^−/− (n=5), Ddit4^+/++ rapamycin (n=5) and Ddit4^−/−+ rapamycin (n=5) mice. Each data point is the mean of ≥10 random field quantified per mouse. Variance is plotted as standard error of the mean with significance estimated by one-way ANOVA with multiple comparisons to the Ifrd^+/+ control (Dunnett post-hoc test).

G. BrdU in pancreas. Boxes: individual proliferating cells. Proliferating cells after rapamycin are stromal, not paligenotic acinar cells. Scale bar, 30 μm; boxed area inset, 15 μm.

H. Data from Ddit4^+/+ (n=5), Ddit4^−/− (n=5), Ddit4^+/++ rapamycin (n=5) and Ddit4^−/−+ rapamycin (n=5) mice as in panel (G) quantified. Datapoint: Mean±SEM of ≥10 random fields quantified per mouse, significance as per panel (D).