DFO induces autophagy. (A) HeLa cells transfected with or without pCMV-Fpn-FLAG were incubated with FAC (10μM Fe) for 24 hours followed by incubation for 10 hours in the absence or presence of 10μM ponasterone A, 100μM DFO, desferasirox, or deferriprone. Cells were incubated with or without chloroquine for 5 hours. Cells were fixed and processed for immunofluorescence using mouse anti-LC3 antibody (green). Nuclei were stained with 4′,6-diamidino-2-phenylindole (blue). The number of LC3B-positive cells was quantified (right panel) and error bars represent the analysis of greater than 5 fields per sample (at least 100 cells). Error bars represent SD from 3 different experiments. (B) HeLa cells expressing Fpn-FLAG were incubated with or without FAC (10μM) for 18 hours in the presence or absence of DFO at different concentrations (0.05, 0.1, 1, 10, 100μM). Cells were fixed and processed for immunofluorescence as in panel A. (C) HeLa cells were incubated with or without FAC (10μM) for 18 hours in the presence or absence of DFO (100μM) or DFO previously saturated with iron. Cells were incubated with or without chloroquine for 5 hours. Cells were fixed, processed for immunofluorescence, and quantified as in panel A.