Fig. 4 | Communications Biology

Fig. 4

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

Fig. 4

Loss of function studies of key ferroptosis regulators in the context of Golgi stress. a Relative survival of control (shLUC) and ACSL4 knockdown HeLa cells following treatment with 35 nM BFA for 72 h; **P < 0.01. ACSL4 knockdown levels are shown by immunoblotting. b Survival ratios of SLC7A11 knockdown and control HeLa cells after treatment for 72 h with either 30 nM BFA or 2 µM erastin; **P < 0.01; ***P < 0.001. SLC7A11 expression is shown by immunoblot analysis. c Relative viability of GPX4 knockdown or control HeLa cells after treatment with 35 nM BFA for 72 h; **P < 0.01. Expression levels of GPX4 are shown by western blot. Protein lysates were run on the same gel, and dashed lines in blots indicate where irrelevant samples were cropped out. ac Cell survival of vehicle-treated and BFA-treated cells was analyzed using the CTB assay. Statistical analysis was performed using Student’s two-tailed t-test. Shown are representative examples of three independent experiments, and three wells per genotype and treatment condition were measured. Center bars indicate the mean, error bars indicate the SD. d Representative immunoblot analysis (n = 2) of HeLa SLC7A11 knockdown (shSLC7A11#1) or control (shRFP) cells treated for 72 h with either vehicle, 40 nM and 50 nM BFA or 1.5 μM GCA. e Representative immunoblot analysis (n = 2) of GPX4-depleted (shGPX4#1) or control HeLa cells treated for 72 h with vehicle, 40 nM and 50 nM BFA or 1.5μM GCA. Note that the same shRFP protein lysates were loaded on separate gels in d and e. f HeLa knockdown cells with the indicated genotypes were treated for 72 h with 40 nM BFA before fixation, GM130 immunofluorescence staining and image acquisition. Graph shows quantification of Golgi area following vehicle or BFA treatment. Center bars indicate the median, and tails indicate the minimum and maximum; boxes extend from the 25th to 75th percentiles. shControls refers to shLUC and shRFP hairpins which were combined for the analysis. On average 1000 cells were analyzed for Golgi area quantification; ***P < 0.001. The corresponding immunofluorescence pictures are presented in Supplementary Fig. 4. g Real-time lipid peroxidation measurements using Liperfluo dye added to vehicle-treated HeLa knockdown cells of the genotypes shown for the indicated amount of time. Each point presented is the average fluorescence intensity derived from three separate wells, and four pictures per well were measured. Error bars indicate SD of the mean. h Relative lipid peroxide fold changes as calculated by dividing the average GFP values (derived from three wells) of HeLa knockdown cells of the indicated genotype treated with 40 nM BFA by their corresponding average GFP values of vehicle-treated cells. Error bars indicate SD of the mean. Statistical significance between the different treatment conditions was calculated using a two-way ANOVA test. GPX4 and SLC7A11 knockdown cell lines (at least one of the two hairpins targeting either GPX4 or SLC7A11) displayed significantly reduced lipid peroxide-fold changes compared to both shRFP and shLUC control knockdown cell lines starting approximately at 24 h (GPX4 knockdown cells) or 30 h (SLC7A11 knockdown cells) of BFA treatment (in comparison to shRFP control cells, significance is reached earlier); P < 0.05. Scanned images of unprocessed blots are shown in Supplementary Fig. 7

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