Fig. 1.

Physapubescin B increases autophagic flux. (A) The chemical structure of physapubescin B. (B) HCT116 (D) HeLa cells were treated with the indicated doses of physapubescin B with or without chloroquine (CQ, 50 μM) for 3 h. Cell lysates were collected and subjected to western blot for detecting LC3 conversion. β-actin was used as a loading control. (C) HCT116 (E) HeLa proteins expression from (B) and (D) was evaluated by ImageJ, means±SD were presented (*, P<0.05; **, P<0.01; ns, P>0.05; Student's t-test). (F) HCT116 cells were treated with physapubescin B (20 μM) for the indicated time points with or without CQ (50 μM). Cell lysates were collected and subjected to western blot for detecting LC3 conversion. β-actin was used as a loading control, and (G) the proteins expression was evaluated by ImageJ, means±SD were presented (**, P<0.01; ns, P>0.05; Student's t-test). (H) HeLa cells stably expressing GFP-LC3 were incubated with the indicated concentrations of physapubescin B with or without CQ (50 μM) for 3 h. Autophagosomes were inferred by the presence of GFP-LC3 puncta under confocal microscopy (×600). Scale bar, 5 µm. Amino acid starvation (AA-) was used as a positive control for induction of autophagy. (I) The mean fluorescence intensity (MFI) was analyzed by ImageJ, means±SD were presented (*, P<0.05; **, P<0.01, Student's t-test).