Fig. 7.

Role of autophagy in HMA response. (A) ARPE-19 cells were treated with HMA in the absence or presence of 1 μM rapamycin or 5 mM 3-MA. After 2 or 4 hrs of treatment, cells were trypsinized and counted. A total of 1000 cells were seeded into 6-well plates. Seven days after seeding, cells were stained with cresyl violet. Controls using rapamycin or 3-MA alone have show no difference with the control (not shown) (B) Using image analysis, the surface covered by the cells was measured and plotted. For 2 hrs of treatment all means were significantly different (P < 0.001) for 4 hrs no significant difference was found between control and HMA+3-MA treated cells (student test P = 0.73) or between HMA and HMA + rapamycin-treated cells (P = 0.87). The treatment of cells with rapamicin or 3-MA alone did not have any effect on cell survival (not shown). (C) HMA-treated ARPE-19 cells, at different concentrations for 24 hrs, were stained with DAPI (upper panel) and anti-p62 antibody (lower panel). HMA-treated samples were compared to 50 nM bafylomicin-treated (representative of lysosome-autophagosome fusion failure) or 1 μM rapamycin (representative of autophagy)-treated cells. Scale bar 25 μm.