Analysis of autophagic flux and lysosomal staining in adck3 mutant cells in response to antioxidant and Cyclosporine A treatment.

(A, B). Analysis of autophagic flux in control and adck3 mutant fibroblasts. GFP-LC3 transduced cells (A) were analysed via fluorescence microscopy for the appearance of GFP-LC3 puncta. RAPA– 1 mM Rapamycin treatment. WCEs (15 μg) from each cell line with or without bafilomycin A treatment (Baf) were also analysed via immunoblotting (B) with anti-LC3B and anti-GAPDH. Images were acquired and quantified using the Licor Odyssey IR based platform. I: LC3 Isoform I; II: LC3 Isoform II. Results displayed as LC3II/GAPDH ratio normalised to control values without bafilomycin treatment ± S. E. M. n = 3. = p ≤ 0.05 and = p ≤ 0.005 using Student’s t-test (compared to control) in all histograms. (C, D). Control and adck3 mutant fibroblasts were incubated with DMSO, 0.5 mM N-acetyl cysteine (NAC), 10/100 nM mito-Q or 5 μM Cyclosporine A for 6 days in complete media prior to analysis using lysotracker red and flow cytometry. Media was changed every 2 days with the addition of fresh compound/s. n = 2. Statistical analysis by Student’s t-test showed no significant changes.