Fig. 4: FABP5 biomarker validation in RSL3-treated and GPX4 knockout cells.

A FABP5 fluorescence intensity changes after RSL3 or GPX4 knockout (KO) in cell lines of different etiologies. Calu-1, HCC827, HT-1080 and U-138MG cells after treatment with RSL3 (200 nM, 5 h), and SH-SY5Y and human fetal fibroblast (hFF) cells with RSL3 (200 nM) + ammonium ferric citrate (FAC, 250 μM) for 3 h. 2 × 10³ cells were assayed per condition. RSL3 final intensity is shown as mean ± SD of n = 3 independent experiments with three replicate wells each. Fold change at 96 h post infection with GPX4 KO or control (empty vector) lentivirus is shown as mean ± SD of at least three independent experiments with 2 × 10³ cells assayed per condition in each of three replicate wells. SH-SY5Y and hFF are viable following GPX4 KO-induced lentivirus. B Live cell brightfield and fluorescence images of HT-1080 viability (indicated by DAPI penetration) were taken hours post infection (p.i.) with GPX4 KO and control virus at 72 and 96 h. Increased loss of viability was observed at 96 h, while early detection of FABP5 antigen at 72 h is shown in C when only a fraction of cells have died. C Western analysis of respective proteins in HT-1080 cells 72 h following GPX4 ablation. Statistics were calculated using two-way ANOVA (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns not significant).