Figure 4.

Morphine-mediated defective autophagy involves upstream activation of ER stress in human astrocytes. (A-D) Representative western blots showing the protein levels of BECN1 (A), LC3-II (B), p62 (C) and GFAP (D) in human A172 astrocytoma cell line pretreated with 100 nM wortmannin for 1 h followed by 500 nM morphine for 24 h. (E) qPCR showing relative expression of proinflammatory cytokines such as TNF and IL6 mRNA, and (F) Representative western blot showing the expression of the BiP protein in human A172 astrocytoma cell line pretreated with 100 nM wortmannin for 1 h followed by 500 nM morphine for 24 h. (G-K) Representative western blots showing the protein levels of BECN1 (G), LC3-II (H), p62 (I), BiP (J) and GFAP (K) in human A172 astrocytoma cell line transfected with BECN1 siRNA and scrambled siRNA followed by 500 nM morphine for 24 h. (L) qPCR showing the relative expression of proinflammatory cytokines such as TNF and IL6 mRNA in human A172 astrocytoma cell line transfected with BECN1 and scrambled siRNAs followed by 500 nM morphine for 24 h. (M-Q) Representative western blots showing protein levels of BECN1 (M), LC3-II (N), p62 (O), BiP (P) and GFAP (Q) in human primary astrocytes transfected with BECN1 and scrambled siRNAs followed by 500 nM morphine for 12 h. (R) qPCR showing the relative expression of proinflammatory cytokines such as TNF and IL6 mRNA in human primary astrocytes transfected with BECN1 and scrambled siRNAs followed by 500 nM morphine for 12 h. β-actin was used as a loading control for western blot and GAPDH for mRNA expression of cytokines. Data are presented as mean ± SEM; n = 6. Non-parametric Kruskal-Wallis One-way ANOVA followed by Dunn’s post hoc test was used to determine the statistical significance between multiple groups: *, P<0.05 vs. control; #, P<0.05 vs. morphine.