Figure 5 . Ziyu II induces autophagy by repressing the Akt/mTOR pathway in colorectal cancer cells both in vitroand in vivo
(A ). Immunobloting of phosphorylation of Akt(S473), mTOR(S2448), p70S6K (S424/T421), and 4EBP1 (S65/T70) in cells treated with the indicated concentrations of Ziyu II for 24h. (B ). DLD-1 and HCT116 cells were transfected with an empty vector (pECE) or with a constitutively active CA-Akt for 48h, and then cells were treated with Ziyu II for another 24h. Akt phosphorylation, and LC3 lipidation were determined by immunoblotting. (C and D ) left, the formation of endogenous LC3 puncta was assessed in cells treated as inB . (E , F ), total number of endogenous LC3 puncta per cell. Scale bars, 10 μm. All data are means±SD. ***, p< 0.001. Akt expression in orthotopic xenografts was examined by IHC. Representative images were provided as indicated (G), and relative immunohistochemical scores were shown (H). Scale bar, 50 μm. ***, p < 0.001.