Fig. 2 | Communications Biology

Fig. 2

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

Fig. 2

Golgi stress leads to ferroptosis. a Real-time lipid peroxidation analysis using the IncuCyte system and Liperfluo staining of HeLa cells treated with vehicle or the indicated concentrations of BFA and erastin (ERS), respectively. Immunofluorescence images of HeLa cells treated for 20 h with 40 nM BFA and stained with Liperfluo are shown on the right; scale bar: 50 µm. Shown is a representative example of three independent experiments. Bars and error bars represent the means and SD, respectively. Statistical significance between the different treatment conditions was calculated using a two-way ANOVA test, and the average fluorescence intensity for each condition was derived from four pictures taken per well from four separate wells. BFA treatment (both concentrations) caused significant differences in lipid peroxide production compared to vehicle treatment starting at ten hours of treatment; P < 0.01. b Schematic illustration of Cys2 uptake/metabolism and GSH biosynthesis and the role of iron and GSH in ferroptosis induction. A number of known ferroptosis/ROS inhibitors are shown (CPX, Fer-1, Lip-1, LOXi, Pran). c Relative viability of a panel of cancer cell lines as well as primary lung fibroblasts after treatment with BFA for 72 h in the presence or absence of 10 µM ferrostatin-1 (Fer-1); **P < 0.01; ***P < 0.001. BFA concentrations used: HeLa: 35 nM; A549, DU145, HT-29 and primary lung fibroblast: 60 nM; Panc-1: 120 nM. d Western blot analysis of HeLa cells that were either vehicle-treated, treated with 35 nM BFA or with 1.5 µM GCA in the presence or absence of 10 µM ferrostatin-1 (Fer-1) for 24 h. β-actin was used as loading control. e, f Relative viability of HeLa cells after treatment with 35 nM BFA or 1.5 µM GCA for 72 h in the presence or absence of 1 µM liproxstatin-1 (Lip-1) (e) or 1 µM CPX (f); ***P < 0.001. g Relative viability of HeLa cells following treatment with 35 nM BFA or 1.5 µM GCA for 72 h in the presence or absence of 10 µM PD-146176 (LOXi); *P < 0.05; ***P < 0.001. h Relative HeLa cell viability in response to 35 nM BFA for 72 h in the presence or absence of 100 µM Trolox, 4 µM NOX1 inhibitor (=GKT137831) or 10 µM pranlukast (Pran); **P < 0.01; ***P < 0.001. c, eh Cell survival was determined using the CTB assay. Statistical analysis was performed using Student’s two-tailed t-test. Shown are representative examples of at least three independent experiments, and three wells per treatment condition were measured. Center bars indicate the mean, error bars indicate the SD. i Western blot analysis of HeLa cells that were treated prior to lysis with the indicated BFA or GCA concentrations for 24 h. j Western blot analysis of HeLa cells incubated in Cys2-free or low-dose (2.5 µM) Cys2-culture medium for 24 h before protein extraction. d, i, j Shown is a representative western blot of three independent experiments. Scanned images of unprocessed blots are shown in Supplementary Fig. 7

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