Extended Data Fig. 3: Cytosolic STARD7 is a suppressor of ferroptosis.

a, Immunoblot analysis of WT cells and WT cells overexpressing FLAG-tagged cyto-STARD7. #1, #2 and #3 represent different monoclonal cell lines selected from a polyclonal population. b, Localization of cyto-STARD7 in WT and cyto-STARD7 overexpressing cells was determined by immunofluorescence in HeLa cells. Cells are stained with antibodies against FLAG (green) to detect overexpressed STARD7 and TOMM20 (red) to detect mitochondria. Immunofluorescence images were taken via confocal microscopy with 1,000x magnification. A merge of both, as well as a magnified images are shown. Scale bars are 10 µm (magnified image). Graph shows the signal intensity plots of STARD7 (green) and TOMM20 (red) through line segment analysis (white line) in magnified image, n = 2 independent experiments. c, Cell death measured in WT and cyto-STARD7 overexpressing monoclonal cell lines treated either with erastin (3 µM) (n = 3) or PUFA (arachidonic acid 20:4, 40 µM) (n = 2), or combination of erastin and PUFA (n = 3) in the presence of the ferroptosis inhibitor Fer1 (1 µM) or the apoptosis inhibitor QVD (1 µM) as indicated (n = 3). d,e, Total CoQ levels (n = 15 independent biological samples) (d) and CoQ synthesis (e) measured in WT and cyto-STARD7 overexpressing cells incubated with ¹³C₆ glucose for the indicated time points. f, Ferroptosis was induced in WT and cyto-STARD7 overexpressing cells by either inhibiting GPX4 arm via erastin (1 µM) and PUFA (40 µM) or by inhibiting both GPX4 and CoQ-FSP1 arm via combination of erastin+PUFA and iFSP1 (1 µM). Cell death was measured after 24 h. a, n = 2 and f, n = 3 independent experiments and e, n = 5 (with one blank at 0 h from each genotype) biologically independent samples. c−f, Data is represented by 95% confidence interval of the mean and the P values were calculated using a two-tailed Student’s t-test for unpaired comparisons where indicated.

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