SPC stimulates mTOR via the Akt signaling pathway in Mel-Ab cells. Cells were preincubated with 20 μM LY294002 for 30 min prior to the addition of 10 μM SPC and then incubated for another 3 d. (A) Cells were lysed and the levels of LC3 II were analyzed by Western blotting. Actin was used as a loading control. (B) Melanin content was measured as described in the Materials and Methods. Data represent means ± SD of triplicate experiments. ** P < 0.01 compared to the SPC-treated cells. (C) After serum starvation for 24 h, cells were incubated with 10 μM SPC for 30 min in the presence or absence of 20 μM LY294002. Whole cell lysates were analyzed by Western blotting with antibodies against phospho-Akt, Akt, phospho-mTOR, mTOR, and actin (loading control). Fold increases over the control were determined by densitometric analysis and are shown below each lane.