Fig. 2: Both (R)-CRZ and (S)-CRZ are specific inhibitors of ferroptotic cell death.

A (R)-CRZ and (S)-CRZ inhibited RSL3-induced ferroptosis in HT-1080 cells. Fer-1 (1 μM), (R)-CRZ (1 μM) and (S)-CRZ (1 μM) were used in the experiment. Cell death was detected by PI staining and FACS analysis at 8 h after treatment. B (R)-CRZ and (S)-CRZ inhibited RSL3-induced ferroptosis in SK-HEP-1 cells. The experiment was performed with the same conditions and analysis method used in A. C Chemical structures of (R)-CRZ and (S)-CRZ. D (R)-CRZ and (S)-CRZ have similar inhibitory effect on ferroptosis. EC₅₀ of (R)-CRZ and (S)-CRZ against ferroptosis induced by RSL3, erastin or FIN56 in HT-1080 cells are indicated. For RSL3 treatment, 1 μM of drug was used and cell viability was measured at 10 h after treatment. A concentration of 10 μM of erastin was used and cell viability was measured11 h after treatment. For FIN56, 5 μM of drug was used to induce ferroptosis and cell viability was measured at 17 h after treatment. E (R)-CRZ (3 μM) and (S)-CRZ (3 μM) inhibited spontaneous ferroptosis in GPX4 KO monoclonal HT-1080 cell lines. Cell death was measured after 10 h of treatments. F (R)-CRZ (3 μM) and (S)-CRZ (3 μM) do not protect HT-1080 cells from H₂O₂ (3 mM)-induced necrosis. Cell viability was measured at 8 h after treatment. Data are presented as the mean ± S.D., n = 4 independent repeats. G (R)-CRZ (3 μM) and (S)-CRZ (3 μM) did not protect HT-1080 cells from STS-induced apoptosis. z-VAD, a known apoptosis inhibitor, used as a positive control at 50 μM to inhibit apoptosis. Cell viability of HT-1080 was tested at 16 h after drug treatment. STS (12 μM) was used in 786-O cell line for 12 h to induce apoptosis. H (R)-CRZ (3 μM) and (S)-CRZ (3 μM) did not protect 786-O cells from ABT-263 (20 μM)-induced apoptosis. 50 μM of z-VAD was used as a positive control at to inhibit apoptosis. Cell viability was tested at 8 h after drug treatment. I Other MTH1 inhibitors (TH588 and TH287) do not suppress ferroptosis. Cell viability was tested at 10 h after drug treatment. EC₅₀ of TH588 and TH287 cannot be calculated. J (R)-CRZ and (S)-CRZ inhibit ferroptosis in both ALK⁺ (Karpas-299, SU-DHL-1 and H2228) and ALK^- (HT-1080 and 786-O) cell lines. (R)-CRZ (3 μM) and (S)-CRZ (3 μM) were used to treat cells with different concentration of RSL3. Because the measurements in some groups did not reach the plateau, those EC₅₀ calculations represent approximate values. Data in A, B, D, E, G–J are presented as the mean ± S.D., n = 3 independent repeats.