Journal: Communications Biology

Article Title: Golgi stress mediates redox imbalance and ferroptosis in human cells

doi: 10.1038/s42003-018-0212-6

Figure Lengend Snippet: ROS induction and depletion of glutathione in response to Golgi stress. a Analysis of intracellular reactive oxygen species (ROS) levels using dihydroethidium (DHE) staining and flow cytometry of HeLa cells treated for 48 h with the indicated compounds. CCCP was used as a positive control. b Quantification of the relative DHE mean fluorescence intensity (MFI) levels of samples shown in the FACS histogram in a ; ** P < 0.01; a.u. arbitrary unit. c Quantification of total intracellular glutathione (GSH) levels in HeLa cells treated with 60 nM BFA or 2 µM GCA for 48 h; ** P < 0.01. d Relative viability (survival of compound-treated cells divided by survival of vehicle-treated cells) of HeLa cells after treatment with BFA (35 nM), AMF-26 (35 nM) or GCA (1.5 µM) for 72 h in the presence or absence of glutathione (GSH, 2 mM) or N -acetyl cysteine (NAC, 1 mM) as measured by the CellTiter-Blue (CTB) assay; * P < 0.05 and ** P < 0.01. a – d ) 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. e Displayed is the relative mean cell viability ±SD of HeLa cells stably overexpressing Flag-γ-Tubulin, Flag-GSR or Flag-GSTA1 following treatment with 35 nM BFA for 72 h; ** P < 0.01. Cell survival was measured by the CTB assay. Shown is a representative example of three independent experiments, and three wells per genotype and treatment condition were measured. Expression levels of overexpressed proteins are shown by immunoblotting. Protein lysates were run on the same gel, and dashed lines in blots indicate where irrelevant samples were cropped out. a – e Statistical analysis was performed using Student’s two-tailed t -test. Scanned images of unprocessed blots are shown in Supplementary Fig.

Article Snippet: Indicated chemicals were obtained from companies (in parentheses): brefeldin A (Sigma-Aldrich), golgicide A (Sigma-Aldrich), AG1478/tyrphostin (Sigma-Aldrich), Erastin (MedChem Express), ferrostatin-1 (Sigma-Aldrich), liproxstatin-1 (Sigma-Aldrich), NOX1/4 inhibitor (GKT137831, Cayman Chemical), Sulfasalazine (Sigma-Aldrich), Sorafenib (Sigma-Aldrich), RSL3 (MedChem Express), Glutathione (Sigma-Aldrich), N -acetyl-cysteine (Sigma-Aldrich), Ciclopirox olamine (CPX, Sigma-Aldrich), LOXi (PD-146176, Santa Cruz Biotechnology), Trolox (Sigma-Aldrich), Prankulast (Biomol), tunicamycin (Sigma-Aldrich), erastin (MedChemExpress), BSO (Santa Cruz Biotechnology), cisplatin (Santa Cruz Biotechnology), doxorubicin (Sigma-Aldrich), 2-ME (Sigma-Aldrich), PPG (Sigma-Aldrich), Nutlin-3 (Sigma-Aldrich), vildagliptin (Sigma-Aldrich), Q-VD-OPh hydrate (Sigma-Aldrich), bafilomycin A (Sigma-Aldrich), nocodazole (Sigma-Aldrich), panobinostat (Sigma-Aldrich), doxorubicin (Sigma-Aldrich).

Techniques: Staining, Flow Cytometry, Positive Control, Fluorescence, CtB Assay, Stable Transfection, Expressing, Western Blot, Two Tailed Test