Figure 7. Rapamycin-mediated activation of autophagy enhanced the clearance of Mut-TGFBI. At 60–80% confluence, cells were treated with inhibitors for 14 h. (A) Cells were treated with either rapamycin or DMSO, and extracts were probed with anti-TGFBI and anti-LC3 antibodies. Rapamycin (Rap) decreased the levels of mut-TGFBI in HO corneal fibroblasts. Representative blots are shown of samples from three independent experiments. (B) Autophagy was measured as the ratio of LC3-II/ACTB from immunoblots shown in (A) following normalizing of protein levels to ACTB using densitometry. (C) Quantification of the TGFBI levels in (A). Baf A₁ (0.1–0.2 μM) and Rap (100 nM) were added to the medium where indicated. (D) WT and GCD2 HO corneal fibroblasts were treated with Rap (100 nM) and Baf A₁ (0.1 μM) individually and in combination for 14 h. Cells were lysed and immunoblotted with anti-LC3 (first panel), anti-TGFBI (second panel) or anti-ACTB (third panel) antibodies. Autophagy was measured as the ratio of LC3-II/ACTB and is represented as the fold increase in this ratio relative to WT untreated corneal fibroblasts. (E) The ratio of LC3-II/ACTB shown in (D). (F) Quantification of the TGFBI levels shown in (D). Results represent the mean ± SD (n = 3). *p < 0.05; **p < 0.01; NS, not significant.