Fig. 6.

Regulation of ferritinophagy in HT1080 cells. A. Primary human macrophages and HT1080 cells were incubated for 24 h under hypoxia: FTMT, FTH, and NCOA4 were analyzed by Western blotting. For quantification, intensities were normalized to lane normalization factor (LNF) (n = 3). B–C. HT1080 cells were transfected with a siRNA against NCOA4 and Western analysis was performed for FTMT and FTH. Intensities were normalized to LNF (n = 3). D-E. HT1080 cells were incubated for 24 h under hypoxia and treated with RSL-3 (1 μM) and Liproxstatin-1 (Lip, 1 μM) within the last 4 h of incubation. Western analysis was performed for FTH and FTMT (n = 3). F. HT1080 cells were treated as in D and vitality was assessed by CellTiter blue assay (n = 3). G. HT1080 cells were transfected with a siRNA against FTH and incubated for 24 h under hypoxia followed by CellTiter-Blue assays (n = 4–6). H. HT1080 cells were transfected with a siRNA against FTH and treated with Lip directly after transfection. Vitality was measured by the CellTiter-Blue assay (n = 3). I. Proposed mechanism of ferroptosis in human macrophages vs. HT1080 cells. For details see the text. Complete total protein stains are collected in Fig. S1. All data are depicted with SEM. Students t-test or ANOVA values p < 0.05 were considered as significant.