Fig. 5 | Communications Biology

Fig. 5

From: Golgi stress mediates redox imbalance and ferroptosis in human cells

Fig. 5

Cellular responses to low doses of ferroptosis inducers. a Schematic illustration of Cys2 uptake and GSH metabolism including known ferroptosis inducers such as erastin (ERS) and sorafenib (SRF). bf Relative viability of HeLa cells treated with with BFA (35 nM) or AMF-26 (35 nM) for 72 h in the presence or absence of sublethal doses of erastin (ERS, 1 µM), sorafenib (SRF, 1 µM), sulfasalazine (SAS, 200 µM), L-buthionine sulfoximine (BSO, 50 µM); **P < 0.01; ***P < 0.001. g Shown is the relative survival of HeLa cells grown in medium supplemented with increasing concentrations of Cys2 for 72 h in the presence or absence of 35 nM BFA; **P < 0.01; ***P < 0.001. bg Statistical analysis was performed using Student’s two-tailed t-test. Shown are representative examples of three independent experiments, and three wells per treatment condition were measured. Center bars indicate the mean, error bars indicate the SD. h Real-time analysis of lipid peroxide formation of HeLa cells following BFA treatment in the presence or absence of 1 µM erastin (ERS) using Liperfluo staining. Shown is a representative example of two independent experiments each time measuring the average fluorescence intensity which was derived from four pictures taken per well from five wells. Statistical significance between the different treatment conditions was calculated using a two-way ANOVA test. [BFA + 1 μM ERS] treatment caused significant lower lipid peroxide generation compared to BFA-only treatment starting at 13 h of treatment, whereas 1 μM ERS by itself had no significant effect on oxidation of lipids; P < 0.01. i HeLa cells were treated for 72 h with 40 nM BFA in the presence or absence of ferrostatin-1 (Fer-1, 10 µM), low concentration of erastin (ERS, 1 µM), sorafenib (SRF, 1 µM) or medium containing 7 µM Cys2 (low Cys2) for 72 h. Shown is a representative western blot of three independent experiments. Protein lysates were run on the same gel, and dashed lines in scans indicate where irrelevant samples were cropped out. j Immunofluorescence microscopic pictures of HeLa cells treated for 72 h with vehicle, 1 µM erastin (ERS), 30 nM BFA or a combination thereof; scale bar: 20 µm. k Immunofluorescence microscopic pictures of HeLa cells vehicle-treated, treated with 30 nM BFA, 1 µM sorafenib or a combination thereof for 72 h; scale bar: 20 µm. j, k Quantification of Golgi areas of HeLa cells depicted in the immunofluorescence images on the left is shown in the right graphs. Center bars indicate the median, and tails indicate the minimum and maximum. On average 1000 cells per genotype and conditions were analyzed for the quantification; **P < 0.01. bg Cell survival was determined using the CTB assay. Scanned images of unprocessed blots are shown in Supplementary Fig. 7

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