Fig. 6. Inhibition of the PI3K/Akt pathway stimulated by ursolic acid (UA) plus 3,3’-diindolylmethane (DIM) combination treatment induces Hippo signaling pathway activation in ESCC.
TE-8 and TE-12 cells were pre-treated with LY294002 (50 μM) or 3-MA (5 mM) for 2 h and further treated with UA (20 μM) plus DIM (50 μM) for 48 h. The expression of Hippo pathway-related proteins: (A) Rassf1, Mst1, Mst2, Sav1, Mob-1, p-Mob1, (B) YAP, and p-YAP and Hippo signaling pathway downstream proteins CTGF were examined by Western blotting. (C) Cytoplasm and nuclear proteins were separated to detect YAP distribution by Western blotting. GAPDH was used as the internal control and cytoplasm marker. Lamin B was used as a nuclear marker. (D) TE-8 and TE-12 cells were stained with anti-YAP antibody (green) and DAPI (blue) and visualized under fluorescence microscopy. Nuclei were stained by DAPI. The value of immunofluorescence was measured and calculated by ImageJ software. Data are expressed as the mean ± standard error of the mean. CONT, control; LY, LY294002; Mix, UA plus DIM; ESCC, esophageal squamous cell carcinoma; n.s., no significant. *, compared to the control; a, combined therapy (mix) compared with mix plus LY294002; #, mix compared with mix plus 3-MA. * or #, p < 0.05; **, ## or aa, p < 0.01.
© Korean J Physiol Pharmacol
