Figure 3

Role of CaMKKβ-AMPK-mTOR signaling cascade in Ssd-mediated autophagy. (a) Ssd activates AMPK-mTOR signaling pathways. HeLa and MCF-7 cells were treated with Ssd (10 μM) for the indicated time and analyzed for p-AMPK, p-p70S6K, total p70S6K and actin, respectively. (b) AMPK inhibitor abrogates the Ssd-mediated autophagic effect in cancer cells. HeLa and MCF-7 cells were transiently transfected with the GFP-LC3 plasmid for 24 h and then treated with dimethyl sulfoxide (DMSO; Ctrl) or 10 μM Ssd with or without 5μM of AMPK inhibitor compound C (CC) for 4 h. The cells were then fixed for fluorescence imaging and cell counting. Bar chart represents the quantitation of autophagic cells (percentage of GFP-LC3 dots), error bars, S.D. *P<0.05. (c) CaMKKβ inhibitor abolishes the Ssd-mediated autophagic effect in cancer cells. GFP-LC3-transfected HeLa cells were treated with DMSO (Ctrl) or 10 μM Ssd with or without 25 μM of CaMKKβ inhibitor STO-609 for 4 h. The cells were then fixed for fluorescence imaging and cell counting. Bar chart represents the quantification of autophagic cells, error bars, S.D. **P<0.01. (d) Calcium chelator blocks the Ssd-induced autophagy and diminishes the Ssd-mediated cell cytotoxicity. GFP-LC3-transfected HeLa cells were treated with DMSO (Ctrl) or 10 μM Ssd in the presence of 5 mM of methyl pyruvate (MP) or 10 μM of calcium chelator BAPTA/AM (BM) for 4 h. The cells were then fixed for fluorescence imaging and cell counting. Bar chart represents the quantification of autophagic cells. The viability of HeLa cells treated with indicated concentrations of Ssd with or without 5 μM BM were measured by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay. Data are the means of three independent experiments; error bars, S.D. **P<0.01; ***P<0.001. (e) Schematic diagram to illustrate the Ssd-induced autophagy via CaMKK-AMPK-mTOR signaling cascade