Figure 6
WWOX suppresses autophagy following MTX treatment. (a) SCC-9 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 cell lysates were examined for LC3 protein expression using western blotting. β-Actin was used as a loading control. (b) SCC-15 cells were infected with empty lentivirus or lentivirus expressing WWOX shRNA. Two days after infection, SCC-15 cells were treated with or without 10 μg/ml MTX and cultured for an additional 24 h. Total cell lysates were examined for LC3 protein expression using western blotting. β-Actin was used as a loading control. (c) SCC-9 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 48 h. Total cell lysates were examined for the expression of Atg12–Atg5 protein conjugate using western blotting. β-Actin was used as a loading control. (d) SCC-9 and SCC-15 cells were transfected with a vector encoding GFP-WWOX or a control GFP vector by electroporation. After culturing for 24 h, total cell lysates were analyzed for Beclin-1 protein expression using western blotting. β-Actin was used as a loading control. (e) Diagrams illustrating the potential effects of WWOX on MTX susceptibility. Left, elevated expression of WWOX renders SCC cells susceptible to MTX-induced apoptosis by suppressing autophagy. Right, SCC cells counteract MTX-induced metabolic stress by activating autophagy. Absent expression of WWOX is associated with resistance to chemotherapeutic drugs in cancer cells
