Fig. 4
Generation and characterization of 4T1 xCTKO cell clones. A Western blot analysis of xCT expression, using vinculin as loading control. B Selenocystine uptake assay (used as a surrogate of cystine uptake), expressed as % fluorescence emitted as compared to parental 4T1 cells. Cells incubated with SAS or Erastin are used as control. C Cell proliferation curves of parental 4T1 cells, WT and KO clones, assessed by MTT assay. D Left: Colony-forming efficiency assay. Right: Percentage of plate surface occupied by colonies. E Left: FACS analysis of ROS content following 4 h incubation with 100 µM of tBHP, indicated by 2’,7’-Dichlorofluorescin diacetate (DCF-DA) fluorescent signal. Right: DCF fluorescent signal represented as fold change of DCF MFI, normalized on cells incubated in growth medium alone. F Right: Representative images of migrating cells in a wound-healing assay at 0 and 48 h post scratching. Left: Percentage of wound closure (of initial wound area) at 48 h. G Left: Percentage of transwell area covered by migrated cells. Right: Representative images of migrating cells in a transwell migration assay. Number of replicates: each dot represents an independent biological replicate, which is the result of at least two technical replicates, except for experiments of flow cytometry, where only a technical replicate for biological sample was performed. Statistical analysis: unpaired t test (panels C-F, G) or ratio paired t test (panels B, E). *p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001. Where not indicated, p value is not significant, except in panel B, where dots and lines depicting comparisons with negative controls are omitted for a better visualization. Lines (panel C only) and histograms represent mean values. Error bars are shown only when n > 5, and represent SD