Fig. 2

Knockdown of GPX4 and indirect inhibition of GPX4 activity by RSL3 trigger ferroptosis in AML cells. (A) MOLM-13 and MV4-11 cells were transduced with two specific shRNAs for GPX4 (sh-GPX4) or negative control (sh-NC) to inhibit GPX4 expression, and the protein levels of GPX4 were measured. (B) Viability was measured in MOLM-13 and MV4-11 cells after transduction with sh-GPX4 or sh-NC for 24, 48, and 72 h. (C and D) Lipid ROS levels were measured by flow cytometry in MOLM-13 and MV4-11 cells with knockdown of GPX4 or NC. Representative plots (left) and statistical analysis of lipid ROS levels (right) are shown. (E) Relative PTGS2 levels were measured in MOLM-13 and MV4-11 cells after transduction with sh-GPX4 or sh-NC. (F) Cell viability was measured by CCK-8 assay in MOLM-13 and MV4-11 cells treated with the indicated concentrations of RSL3 for 24 h, and the IC50 was calculated. (G and H) Cell proliferation and viability were measured in MOLM-13 and MV4-11 cells treated with or without RSL3 (0.1 µM) for 24 and 48 h. (I and J) Lipid ROS levels were measured in MOLM-13 and MV4-11 cells treated with RSL3 (0.1 µM) for 24 and 48 h. Shown are the representative plots (left) and statistical analysis of lipid ROS levels (right). (K) MDA amounts were measured in MOLM-13 and MV4-11 cells treated with or without RSL3 (0.1 µM) for 24 and 48 h. (L and M) Viability was measured in MOLM-13 and MV4-11 cells, which were preincubated with Fer-1 (2 µM), Z-VAD (20 µM), Ac-DEVD (20 µM), CQ (10 µM), or Nec-1 (50 µM) for 1 h and then treated with RSL3 (0.1 µM) for 24 h. **P < 0.01; ***P < 0.001 versus negative control or untreated cells. N.S (not significant) compared with RSL3 treatment