Fig 1.
PP7 inhibits the proliferation of HepG2 cells.
(A) Cells were treated with indicated concentrations of PP7 for 24, 48 or 72 h. The cell viability was analyzed by MTT assay. Values represent the mean ± SD of at least three independent experiments. ** P < 0.01, versus control groups. (B) Morphology of HepG2 cells treatment with PP7 or vehicle control for 24 h was observed under light microscopy (10X objective). Scale bars represent 250 μm.
Fig 2.
Induction of autophagy by PP7 in HepG2 cells.
(A) Monodansylcadaverine (MDC) and (C) Acridine orange (AO) staining. HepG2 cells were treated with vehicle or PP7 for 24 h, stained with MDC and AO, and observed under InCell 2000 confocal microscope (20X objective). The mean fluorescence intensities of MDC (B) and AO (D) in the cells were calculated. (E) GFP-LC3-transfected HepG2 cells were incubated with or without PP7 for 24 h. The GFP-LC3 puncta was observed by InCell 2000 confocal microscope (60X objective). (F) Quantification of autophagic cells (E). Scale bars represent 20 μm. Western blot analysis of the LC3II, Beclin-1 and P62 in HepG2 cells treated with 1.32 μM PP7 for different times (G) or treated with varying concentrations of PP7 for 24 h (H). (I) and (J) were densitometic analysis of (G) and (H), respectively. Data are represented as means ± SD from 3 independent experiments. * P < 0.05, ** P < 0.01, versus control groups.
Fig 3.
Inhibition of autophagy decreased PP7-induced cell death and apoptosis in HepG2 cells.
(A) HepG2 cells were pretreated with 10 μM chloroquine (CQ) for 1 h prior to treatment with 1.32 μM PP7 24 h. The inhibitory effects of CQ on protein levels of LC3II and cleaved caspase-3 were determined by Western blotting. (B) and (C) Densitometic analysis of (A) from three experiments. (D) Pretreatment with 10 μM CQ for 1 h prior to treatment with 1.32 μM PP7 for 24 h. Cell viability was evaluated by MTT assay. Values represent the mean ± SD of at least three independent experiments. ** P < 0.01, compared to PP7-treated alone groups.
Fig 4.
Effects of PP7 on the expression levels of components of PI3K/AKT/mTOR and JNK signaling pathways.
HepG2 cells were treated with 1.32 μM PP7 for different time points (A) or the cells were treated with varying concentrations of PP7 for 24 h (B). Levels of total and phosphorylated proteins were determined by Western blot. (C), (E), (D) and (F) were densitometic analysis of (A) and (B) from three experiments, * P < 0.05 and ** P < 0.01, versus control.
Fig 5.
The role of JNK in PP7-triggered autophagy in HepG2 cells.
(A) HepG2 cells were incubated in 1.32 μM PP7 for 24 h with or without pretreatment of JNK inhibitor SP600125 (20 μM, 1 h). The protein levels of P-JNK, JNK, P62, Beclin-1 and LC3II was detected by Western blot. (B), (C), (D) and (E) were densitometic analysis of (A) from three experiments. MTT assay was used to evaluate the viability of HepG2 cells treated by 1.32 μM PP7 for 24 h with or without pretreatment of 20 μM SP600125 for 1 h (F). Values represent the mean ± SD of at least three independent experiments. ** P < 0.01, compared to PP7-treated alone groups.
Fig 6.
Schematic diagram showing the proposed autophagic signaling pathways triggered by PP7 in HepG2 cells.