Figure 4.

p62 up‐regulation occurs independently of autophagy. Hep3B cells were treated with vehicle (MeOH), EFV (10 or 25 μM), Tg 2 μM or the inhibitor of autophagy 3‐methyladenine (3MA) 2.5 mM for 24 h. (A) Representative Western blot image of LC3 and summary of densitometry data after normalization with the expression of β‐Actin in cells transiently transfected with siControl or siSQSTM1 where the expression of LC3‐II in untreated siControl cells was considered 100%. The absence of p62 confirmed the efficacy of SQSTM1 silencing. (B) Relative mRNA levels of SQSTM1, in absence or presence of 2.5 mM 3MA, were analysed by quantitative RT‐PCR and normalized versus the housekeeping gene β‐Actin (ACTB). (C) Relative mRNA levels of SQSTM1, in siControl and siATG5 cells, were analysed by quantitative RT‐PCR and normalized versus the housekeeping gene β‐Actin (ACTB). Data (mean ± SEM, n = 5) were calculated as percentage of control (untreated cells). *P < 0.05, for efavirenz and #P < 0.05 for thapsigargin, significantly different from vehicle; one‐way ANOVA followed by a Newman–Keuls test. n.s., no significant effects of siRNA or 3MA; Student's t‐test. (D). The expression of ATG5 in order to verify its silencing was studied by Western blotting (representative image) and the expression of GAPDH was employed as a reference.