Figure 3.

BBC3-mediated autophagy processes in U937-differentiated macrophages exposed to silica. (a and b) Macrophages were pretreated with or without BafA1 (50 nM) (b) for 1 h followed by SiO₂ treatment for 12 h. Representative Western blot and densitometric analyses showing that bafilomycin A1 (BafA1) further increased accumulation of LC3BII and SQSTM1 induced by SiO₂. Data are presented as the mean±SEM (n=3); *P<0.05; **P<0.01; ***P<0.001 versus the control group; ^#P<0.05 versus the SiO₂ group (Student's t-test). (c and d) Representative Western blot and densitometric analyses showing the effects of Bbc3-specific siRNA on the expression of BECN1 and LC3B induced by SiO₂. The results suggest that SiO₂ induced BECN1 and LC3BII expression, but reduced SQSTM1 expression, which were reversed by the Bbc3-specific siRNA. Data are presented as the mean±SEM (n=3); *P<0.05; **P<0.01; ***P<0.001 versus the con-siRNA group; ^#P<0.05 versus the con-siRNA+SiO₂ group (two-way ANOVA). (e) U937-differentiated macrophages were transfected for 12 h with fluorescent mRFP-GFP-tagged LC3 plasmids and then treated with Bbc3-specific siRNA or non-specific siRNA using Lipofectamine 2000, followed by treatment with SiO₂ for 24 h. Images were captured by confocal microscopy. The numbers of red, green, and yellow puncta were analyzed to evaluate autophagic flux, and more than 10 cells were quantified for each condition. SiO₂ significantly induced autophagy (yellow and red puncta), but Bbc3-specific siRNA reversed this effect. Scale bar=10 μm. Images are representative of five independent experiments