Fig. 4: Cell death commitment upon GPX4 inhibition and dependencies on WEHI-539 for cell survival.

A Cells were treated with WEHI-539 (10 µM) and proliferation was observed for 7 days, both in the presence and absence of RSL3 (100 nM). Supernatants were changed every second day. Data are mean ± SD of technical triplicates from one representative of 2 independent experiments. B–D The scheme (B) illustrates the 24 h treatment schedule with RSL3 (100 nM) and WEHI-539 (10 µM) or ferrostatin-1 (FER, 2 µM), followed by PBS washing. Cell death was measured by PI uptake via flow cytometry after 6 h. Data are from one representative of three independent experiments. E Cells were subjected to prolonged treatment with RSL3 (100 nM) and WEHI-539 (10 µM) for 16 days (U87 cells) or 22 days (Pfa1 cells), then washed with PBS and re-exposed as indicated. Cell death was determined by PI uptake. Data shown are mean ± SD of technical triplicates from one representative of two (Pfa1) or three (U87) independent experiments. F–J Scheme (F) displays the treatment schedule. Cells received a 1st treatment with DMSO or RSL3 (100 nM) for 2 h (G) or 4 h (I). Cells were then washed and exposed to either FER or WEHI-539 (H, J). Cell death was measured by PI uptake after the first treatment as well as 6 h and 24 h after the medium change. One representative of two independent experiments is shown.