Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) protects hepatocytes from thapsigargin-induced apoptosis.

(A) HCV NS5A inhibits PARP cleavage in HepG2 cells. Western blot analysis shows the expression of PARP and cleaved PARP in HepG2 control and HepG2-NS5A cells treated for 24 h with or without thapsigargin (1 µM). Blots were reprobed with GAPDH-specific antibodies to assess equivalent protein loading. (B) HCV NS5A inhibits caspase-3 expression in HepG2 cells. Western blot analysis shows the expression of procaspase-3 and caspase-3 in HepG2 control and HepG2-NS5A cells treated for 24 h with or without thapsigargin (1 µM). (C) HCV NS5A inhibits the caspase-3/-7 activity in HepG2 cells. The Caspase-Glo 3/7 assay (Promega, Madison, WI, USA) shows the caspase-3/-7 activity in HepG2 control and HepG2-NS5A cells treated for 6 h with or without thapsigargin (0.5 µM). Data are expressed as mean ± standard deviation. *P<0.05. (D) HCV NS5A inhibits caspase-7/-9 and enhances cellular FADD-like interleukin-1beta-converting enzyme (FLICE)-like inhibitory protein, long form (c-FLIP_L) expression in HepG2 cells. Western blot analysis shows the expression of procaspase-7 and caspase-7 (upper panel), procaspase-9 and caspase-9 (middle panel) and c-FLIP_L and c-FLIP, short form (c-FLIP_S), in HepG2 control and HepG2-NS5A cells treated for 24 h with or without thapsigargin (1 µM). Blots were reprobed with tubulin-specific antibodies to assess equivalent protein loading. (E) HCV NS5A enhances XIAP expression and inhibits Bax expression in HepG2 cells. Western blot analysis shows the expression of XIAP, Bax, Bcl-xl, Bcl-2 and p53 in HepG2 control and HepG2-NS5A cells treated for 24 h with or without thapsigargin (1 µM). Blots were reprobed with GAPDH-specific antibodies to assess equivalent protein loading. Densitometric analyses were performed using ImageJ software.