Fig. 6.

Metformin promotes autophagic flux at the autophagosome-lysosome fusion level. A, B, and C, The expression levels of LC3 and p62 in Ang II-induced senescent VSMCs were examined by immunoblot. VSMCs underwent incubation in the presence or absence of Ang II (2 μM) for 24 h, with metformin (200 μM) treatment or not. GAPDH was utilized for normalization (one-way ANOVA, Tukey post hoc; n = 3). D and E, To inhibit autophagy, VSMCs were incubated with bafilomycin A1, then treated with metformin and Ang II; LC3 protein levels were assessed by immunoblot, with GAPDH as a loading control (one-way ANOVA, Tukey post hoc; n = 5). F and G, AMPK protein, and phosphorylation levels were evaluated by immunoblot in senescent primary VSMCs treated or not with metformin. H, To evaluate metformin’s effect on autophagic flux, autolysosome staining was performed; scale bar = 100 μm. I, VSMCs was pre-incubated with bafilomycin A1 for two hours, then treated with metformin and Ang II; LC3, p62, p53, and p21 protein levels were evaluated by immunoblot. J-M, LAMP1, CSTL, CSTB protein levels were examined by immunoblot (one-way ANOVA, Tukey post hoc; n = 3–4). Data are mean ± SD. *P < 0.05, ^**P < 0.01, and ^***P < 0.001 versus control group or for indicated comparisons. Baf A1, bafilomycin A1.