Fig. 7

Autophagy inhibition leads to dysfunctional mitochondria and apoptosis. a Representative western blot of mitophagy markers from D2.0 R cells plated on BME or BME plus COL1 with or without HCQ on days 1 and 5. b Representative images of live D2.0 R cells transfected with the GFP-LC3 reporter and stained with MitoTracker® Red CMXRos on BME or BME plus COL1 matrices for 5 days. Scale bar is 20 µm c Mitochondrial (MitoTracker® Green), mitochondrial reactive oxygen species (ROS; MitoSox™) and mitochondrial membrane potential (TMRM) quantification in D2.0 R cells on BME or BME plus COL1 matrices with or without HCQ (mean ± s.e.m, n = 60,000 cells from 3 independent experiments. Comparisons by Mann–Whitney U-test, two-sided. **P ≤ 0.01; ****P ≤ 0.0001). NT, non-treated; HCQ-D5, hydroxychloroquine treatment beginning on day 5; MFI, mean fluorescence intensity. d MitoTempo is protective of ROS-induced cell death in D2.0 R cells on BME treated with HCQ. Cells were pre-treated for 5 days with 20 μM MitoTempo and subsequently exposed to 50 μM HCQ for 24 hrs. Cell viability was assessed by Cytotox Glo assay (left graph. Mean ± s.e.m, n = 3 wells. Comparisons by unpaired two-sided T-test) and Caspase 3 and 7 activity (right graph. Mean ± s.e.m, n = 3 wells. Comparisons by unpaired two-sided T-test). Data are representative of three independent experiments