Figure 1

Induction of autophagy by saikosaponin-d (Ssd). (a) GFP-LC3 detection of Ssd-mediated autophagy in HeLa and MCF-7 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 5 mM of autophagic inhibitor, 3-MA, for 4 h. Representative micrographs of cells that show GFP-LC3 localization. Bar chart represents the quantitation of autophagic cells. Percentages of autophagic cells demonstrated by the number of cells with GFP-LC3 dots signal (≥10 dots/cell) over the total number of GFP-positive cells in the same field. Arrows represent the cells with GFP-LC3 dots. More than 1000 GFP-positive cells were scored for each treatment. Data are the means of three independent experiments; error bars, S.D. **P<0.01; ***P<0.001 for Ssd-treated cells with and without 3-MA. (b) Direct detection of endogenous LC3-II protein by immunocytochemical analysis. HeLa cells were treated with DMSO (Ctrl) or 10 μM of Ssd for 4 h, then fixed in 4% paraformaldehyde and stained with anti-LC3-II antibodies (red). Images shown are representative of three independent experiments. Arrows indicate the cells with increased endogenous level of LC3-II in cytosolic region. All images are captured under × 60 magnification. (c) Ssd induces autophagic flux. HeLa and MCF-7 cells were treated with Ssd (10 μM) and lysosomal protease inhibitors (10 μg/mL), either alone or in combination, for the indicated time. Cell lysates were analyzed by western blot for LC3 conversion (LC3-I, 18 kDa; LC3-II, 16 kDa) and actin. (d) GFP-LC3 detection of Ssd with or without lysosomal protease inhibitors for 24 h in HeLa and MCF-7 cells. (e) Ssd stimulates the degradation of autophagic substrate p62. HeLa and MCF-7 cells were treated with Ssd (10 μM) for the indicated time. Cell lysates were analyzed using western blot for p62 and actin, respectively. Western blots images were quantified by densitometric analysis from three independent experiments and fold changes include the normalization to actin. Error bars, S.D. *P<0.05; **P<0.01 for cells treated with or without Ssd