Fig. 5

HIF-1α reverses the inhibitory effect of TGF-β/Smad signaling on c-Myc by binding the MH2 domain of Smad3 under hypoxia. a, HIF-1α directly binds the MH2 domain of Smad3. Flag-tagged Smad3 deletion mutants were synthesized in vitro and HA-HIF-1α bound proteins and input were detected by western blotting. b, HIF-1 and p107/E2F4/E2F5 competitively bind the MH2 domain of Smad3. 293T cells were transfected with expression plasmids encoding Flag-tagged genes as indicated as well as HA-Smad3. Co-immunoprecipitation and western blotting were performed. c, Schematic diagram of deletion constructs containing different Smad3 domains. Smad3 domains that bind HIF-1α are shown. d, e, HIF-1α co-localized with Smad2/3 in the nucleus as shown by western blotting (e) and immunofluorescence (d). Co-localization was enhanced by TGF-β treatment (scale bars, 20 μm). f, g, HIF-1α enhanced p-Smad2 and p-Smad3 nuclear localization, which was suppressed by knockdown of HIF-1α, as shown by western blotting (f) and immunofluorescence (g). h, TGF-β promotes the expression of HIF-1α and p-Smad3 in the nucleus. The concentration of all TGF-β used in this part of the experiment was 5 ng/ml