Figure 6

Autophagy enhanced by downregulation of ASPP2 contributes to survival and chemoresistance of HCC cells. (a) HCC-LM3 and HepG2 cells infected with LV-shNon or LV-shASPP2 were treated with EBSS for 6 h with or without 3-MA pretreated 1 h, and then plated in semisolid soft agar medium to monitor anchorage-independent growth. (b) The cells handled as above were plated in dishes for colony assay. (c) HCC-LM3 and HepG2 cells infected with LV-shNon or LV-shASPP2 were pretreated with 10 mM 3-MA or not for 1 h followed by culture in EBSS for 24 h. The percentage of apoptotic cells was measured by Annexin V-FITC/PI (propidium iodide) double staining. The early apoptotic cells (Annexin V+/PI-) were quantified. (d) The infected HCC cells pretreated with 3-MA for 1 h were seeded in 96-well plates with 5-FU (7.5 μg/ml) or VP16 (30 μg/ml) for MTS assay at 48 and 72 h. Values were given as the means±S.D. of six wells. (e) The cells handled as (d) for 24 h were analyzed by Annexin V-FITC/PI double staining for apoptosis. The early apoptotic cells (Annexin V+/PI-) were quantified. (f) The cells handled as (e) were analyzed by western blotting for conversion of LC3I to LC3II. Data are shown as the means±S.D. from triplicate experiments. (*P<0.05; **P<0.01)