Figure 1: Hypoxia-induced TGF-β secretion contributes to deactivation of Hippo signalling through Zyxin.

(a) MDA-MB-231 cells were cultured in serum-free DMEM under normoxic or hypoxic conditions for 12 h. Culture mediums were collected and active TGF-β1 was measured by Elisa. Student’s t-test was applied. (b) MDA-MB-231 cells were cultured under normoxia or hypoxia for 12 h. TGF-β1 mRNA level was analysed by real-time PCR (RT-qPCR). Student’s t-test was applied. (c,d) MDA-MB-231 cells were treated with 5 ng ml⁻¹ TGF-β or 5 μm ml⁻¹ SB431542 for 12 h, and then collected for western blotting using the indicated antibodies (c) and quantification of Lats2 (normalized to actin) and p-Yap (normalized to Yap) protein levels (d). Ratio t-test was applied. (e,f) Scramble and Zyxin-knockdown MDA-MB-231 cells were treated with 5 ng ml⁻¹ TGF-β for 12 h, and then collected for western blotting using the indicated antibodies (e) and quantification of Lats2 (normalized to actin) and p-Yap (normalized to Yap) protein levels (f). Ratio t-test was applied. (g) Zyxin-knockdown and Yap-knockdown MDA-MB-231 cells were treated with 5 ng ml⁻¹ TGF-β for 12 h. Total RNA was extracted and subjected to RT-qPCR analysis for the indicated genes. Student’s t-test was applied. (h,i) Scramble and Zyxin-knockdown MDA-MB-231 cells were cultured under normoxia or hypoxia for 6 h, and then collected for western blotting using the indicated antibodies (h) and quantification of Lats2 (normalized to actin) and p-Yap (normalized to Yap) protein levels (i). Ratio t-test was applied. All data are mean of n=3 independent experiments. All error bars indicate s.d. *P<0.05; **P<0.01; ***P<0.001.