Induction of autophagy by reversine. (A) OC2 cells were treated without (DMSO) or with reversine (5 and 10 μM) for 24 h. Phase-contrast microscopy were taken with 200× magnification. Autophagic vesicles were observed in cells treated with reversine. (B) OSCC cells were pre-treated with 1 or 3 mM 3-MA for 2 hours, followed by treatment without or with 10 μM reversine. At indicated time points, cells were harvested for analyzing the cytoplasmic form LC3-I or the membrane form LC3-II. (C) OCSL cells were transfected with vectors expressing either GFP or GFP-LC3-II, followed by treatment of 3-MA and/or reversine as described. OSCL cells were analyzed under confocal microscope. (D) The same as (C) except that two different concentrations of 3-MA were evaluated. The percentages of cells with puncta were calculated by examining over 100 GFP positive cells. All values were expressed as means ± SD of three independent experiments, each of which was done in triplicate.