Fig. 3. AS directly targets ACSL4 to inhibit ferroptosis.

(A and B) The cell lysates of HT-1080 (A) or MDA-MB-231 (B) were incubated with different concentrations of Bio-AS for 2 hours and then pulled down by using streptavidin beads. The total proteins (input), bound proteins (PD), and remained proteins (after PD) were immunoblotted as indicated. (C) The cell lysates of HT-1080 were incubated with or without AS for 2 hours and then treated with different concentrations of pronase. The expression of ACSL4 and GPX4 was detected by immunoblotting. (D) The cell lysates of HT-1080 were incubated with or without AS for 2 hours and then treated with increasing melting temperature (37° to 61°C). The expression of ACSL4 was detected by immunoblotting. (E) The recombinant ACSL4 protein was incubated with different concentrations of Bio-AS for 2 hours and then pull-down by using streptavidin beads. The expression of ACSL4 was detected by immunoblotting. (F and G) CRISPR-Cas9–generated ACSL4-knockout HT-1080 cells were transduced with or without human ACSL4 and then treated with AS for 1 hour and stimulated with RSL3. The expression of ACSL4 was detected by immunoblotting (F), and the cell viability was detected by CCK-8 assay (G). (H) Recombinant ACSL4 protein was incubated with different concentrations of AS for 10 min at 37°C. The enzymatic activity of ACSL4 was analyzed by measuring the formation of AA-CoA from AA following the protocol described in Material and Methods. (I) Heatmap of major PE species in WT and ACSL4 KO HT-1080 cells treated with or without AS and then stimulated with RSL3 for 6 hours. Each PE/PC species was normalized to the corresponding mean value. Data are the mean ± SEM, n = 3 biologically independent experiments [(G) and (H)]. Statistical analysis was performed using an unpaired two-tailed Student’s t test. NS, P > 0.05, **P < 0.01, ***P < 0.001. PE, phosphatidylethanolamine; PC, phosphatidyl cholines.