Fig. 7
Nox4 promotes expression of the autophagy marker, LC3B in fibroblasts. (A) ROS-generating activity of Nox4 was targeted by treating CAF1 with a Nox4 inhibitor, GKT137831 (20 μM) or with a H2O2 scavenger, N-acetyl cysteine (N-Ac; 5 μM). After 24 h of treatment, protein lysate was collected and analyzed for the expression of the autophagy markers. (B) Nox4 expression was inhibited with a Nox4-specific siRNA in CAFs. After 24 h of siRNA transfection, CAFs were harvested for western blot analysis to evaluate the levels of beclin-1 and LC3B. (C) RMF were generated to express either a wild type Nox4 or an inactive mutant Nox4 (P437H) under doxycycline-mediated induction (Dox). The inducible RMF (iRMFs) were treated with indicated concentrations of Dox for 24 h prior to cell lysis for western blot analysis. Representative blots from N = 3 independent experiments are shown. (D) CAF viability was determined by PrestoBlue assay following 48 h of chloroquine (CQ) treatment. * represents p < 0.05 vs RMF treated at the same dosage of CQ. (E) GKT137831 (20 uM) reduced viability of CAFs compared to RMF. Fibroblast viability was measured by PrestoBlue reagent using a fluorescence plate reader after 5 days of GKT treatment. # represents p < 0.01 vs RMF. (F) Relative viability of fibroblasts after 48 h of siRNA-mediated knock down of Nox1 and Nox4. # represents p < 0.005 vs non-targeting siRNA (siCon) transfected CAFs and siNox4 transfected RMF. All error bars show SD of N = 3