Figure 5

HB-EGF promotes crypt proliferation under hypoxic stress via EGFR activation and PI3K/Akt and MEK1/2 pathways. (a) Organoid size after exposure to hypoxia for 60 min followed by culture for 1–5 days in the presence of R-spondin 1 and Noggin, with or without HB-EGF (50 ng/ml). (b) Percentage of viable organoids after exposure to hypoxia for 60 min followed by culture for 1–3 days in the presence of R-spondin 1 and Noggin, with or without HB-EGF (50 ng/ml). (c) Representative photomicrographs of crypt-villous organoids cultured in the presence of R-spondin 1, Noggin and HB-EGF (50 ng/ml), plus the addition of: (A, F) no inhibitors; (B, G) AG 1487 (EGFR inhibitor); (C, H) LY294002 (PI3K inhibitor); (D, E, I, J) PD98059 (MEK1/2 inhibitor). Panels A–E represent day 1 of culture; panels F–J represent day 5 of culture. The organoids in panels E and J were exposed to hypoxia (100% nitrogen for 60 min); the organoids in all other panels were exposed to normoxia. (d) Organoid size on day 5 of culture, in the presence of R-spondin 1 and Noggin and HB-EGF, with or without inhibitors, on exposure to hypoxia or normoxia. (e) Quantification of viable organoids on day 1 of culture, in the presence of R-spondin 1 and Noggin and HB-EGF, with or without inhibitors, on exposure to hypoxia or normoxia. NA, medium containing R-spondin 1 and Noggin with no addition of HB-EGF; N, normoxia; H, hypoxia; D1, day 1 of culture; D2, day 2; D3, day 3; D5, day 5. Values represent mean±s.e.m. Two-way ANOVA with Tukey–Kramer pair-wise comparison test.