Fig. 3: JNK1/2 positively regulates ferroptosis via upregulation of ACSL4.

A Erastin treatment induced time dependent phosphorylation of JNK. Cells as indicated were treated with erastin (10 μM for HT1080 cells and 30 μM for B16-F10 cells) for indicated time, western blot images confirm the expression of the indicated protein. B Knocking down of FAK decreases erastin induced phosphorylation of JNK1. B16-F10 cells as indicated were treated with erastin (30 μM) for 9 h, western blot images confirm the expression of the indicated protein. C Knocking down of SRC decreases erastin induced phosphorylation of JNK1. B16-F10 cells as indicated were treated with erastin (30 μM) for 9 h, western blot images confirm the expression of the indicated protein. D Knocking down of JNK1 blocks erastin inducd ferroptosis and lipid peroxidation. B16-F10 cells as indicated were treated with erastin (30 μM) for 14 h for cell death measurement, or 12 h for lipid ROS measurement. Cell death and lipid ROS were measured as described in Fig. 1A. Western blot images confirm the expression of the indicated protein. E Knocking down of JNK2 blocks erastin inducd ferroptosis and lipid peroxidation. B16-F10 cells as indicated were treated with erastin (30 μM) for 14 h for cell death measurement, or 12 h for lipid ROS measurement. Cell death and lipid ROS were measured as described in Fig. 1A. Western blot images confirm the expression of the indicated protein. F Overexpression of JNK1 promotes ferroptosis and lipid peroxidation in HT1080 and B16-F10 cells. Cells as indicated were treated with erastin (10 μM for HT1080 cells and 30 μM for B16-F10 cells) for 12 h for cell death measurement, or 10 h for lipid ROS measurement. Cell death and lipid ROS were measured as described in Fig. 1A. Western blot images confirm the expression of the indicated protein. G Overexpression of JNK2 promotes ferroptosis and lipid peroxidation in HT1080 and B16-F10 cells. Cells as indicated were treated with erastin (10 μM for HT1080 cells and 30 μM for B16-F10 cells) for 12 h for cell death measurement, or 10 h for lipid ROS measurement. Cell death and lipid ROS were measured as described in Fig. 1A. Western blot images confirm the expression of the indicated protein. H Knocking down of JNK1 resultes in downregulation of ACSL4 expression in HT1080. Western blot images confirm the expression of the indicated protein. I Knocking down of JNK1 resultes in downregulation of ACSL4 expression in B16-F10 cells. Western blot images confirm the expression of the indicated protein. J Overexpression of JNK1 promotes the expression of ACSL4 in HT1080 cells and B16-F10 cells. Western blot images confirm the expression of the indicated proteins. K Overexpression of JNK2 promoted the expression of ACSL4 in HT1080 cells and B16-F10 cells. Western blot images confirm the expression of the indicated proteins. L Overexpression of ACSL4 restores the sensitivity to erastin induced lipid peroxidation and ferroptosis of JNK1 KD B16-F10 cells. Western blot images confirm the expression of the indicated proteins in indicated cells. Cells as indicated were treated with erastin (30 μM) for 14 h for cell death measurement, or 12 h for lipid ROS measurement. Cell death and lipid ROS were measured as described in Fig. 1A. M Overexpression of ACSL4 restores the sensitivity to erastin induced lipid peroxidation and ferroptosis of JNK2 KD B16-F10 cells. Western blot images confirm the expression of the indicated proteins in indicated cells. Cells as indicated were treated with erastin (30 μM) for 14 h for cell death measurement, or 12 h for lipid ROS measurement. Cell death and lipid ROS were measured as described in Fig. 1A. Data are derived from three independent experiments, and each value represents the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, t test. (n = 3).