Figure 5
MTX upregulates the autophagy-inhibitor mTOR signaling in SCC-15 cells through WWOX. (a) MTX increased the phosphorylation of mTOR and its downstream p70S6K in SCC-15 cells, whereas suppressed mTOR phosphorylation in SCC-9 cells. SCC-9 and SCC-15 cells were treated with 10 μg/ml MTX for the indicated time intervals. Total protein extracts were prepared and analyzed for the phosphorylation of mTOR and p70S6K at Ser2448 and Thr389, respectively, using western blotting. β-Actin was used as a loading control. (b) By co-immunoprecipitation, WWOX was shown to interact with endogenous mTOR in SCC-15 cells. MTX increased their binding in the cells during treatment of 24 h. (c) SCC-15 cells were transfected with a vector encoding GFP-WWOX or a control GFP vector by electroporation. After incubating at 37 °C for 24 h, cells were treated with or without 10 μg/ml MTX and cultured for an additional 24 h. Total lysates of SCC-15 cells were examined for phosphorylation of mTOR and p70S6K using western blotting. β-Actin was used as an internal control. (d) SCC-15 cells were transfected with WWOXsi or a control scrambled sequence by electroporation. Twenty-four hours after transfection, cells were treated with or without 10 μg/ml MTX. After culturing at 37 °C for 24 h, total cell lysates were examined for protein phosphorylation of mTOR and p70S6K using western blotting. β-Actin was used as a loading control
