Fig 2: Erastin and RSL3 promote the expression of IRP1 and IRP2 in A375 melanoma cells. (A and B) mRNA levels of IRP1 and IRP2 were increased in (A) erastin- or (B) RSL3-treated A375 cells. (C and D) Protein levels of IRP1 and IRP2 were increased in (A) erastin- or (B) RSL3-treated A375 cells. Data are presented as the mean ± SD of three independent experiments. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. IRP, iron regulatory protein.

Fig 3: Overexpression of IRP1 and IRP2 promotes erastin- and RSL3-induced ferroptosis. (A) IRP1 and IRP2 overexpression promoted erastin- and RSL3-induced ferroptotic cell death in A375 melanoma cells. (B) Overexpression of IRP1 and IRP2 increased erastin- and RSL3-induced iron accumulation in A375 cells. (C and D) Overexpression of IRP1 and IRP2 promoted erastin- and RSL3-induced (C) MDA and (D) lipid ROS accumulation. The black line represents the control cells; the red line represents overexpression of IRP1; the green line represents overexpression of IRP2 andthe blue line represents overexpression of IRP1 and IRP2. (E) Protein levels of IRP1 and IRP2 were detected following IRP1 and IRP2 overexpression. Data are presented as the mean ± SD of three independent experiments. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. IRP, iron regulatory protein; MDA, malondialdehyde; HA, hemagglutinin.

Fig 4: Erastin and RSL3 promote the transition of ACO to IRP1. (A) ACO activity decreased significantly in A375 melanoma cells following erastin and RSL3 treatment. (B) Erastin and RSL3 promoted the IRE-binding activity of IRP1. (C) Erastin and RSL3 had no significant effect on the IRE-binding activity of IRP2. The relative abundances of IRP1-Flag and IRP2-Flag were analyzed by RNA immunoprecipitation. Data are presented as the mean ± SD of three independent experiments. ***P<0.001; ****P<0.0001. ACO, aconitase; IRP, iron regulatory protein; IRE, iron-responsive element; sh, short hairpin RNA; n.s., not significant.

Fig 5: Schematic depicting IRP1-mediated ferroptosis in melanoma cells. The Fe3+ transported into cells by TFRC and the Fe2+ released through ferritinophagy are the main sources of iron pools. In wild-type cells, erastin- and RSL3-induced IRP1 promotes the expression of TFRC and suppresses the expression of FPN and ferritin, which increases the intracellular Fe2+ levels and promotes ferroptosis. In the absence of IRP1, these effects are reversed, resulting in significantly decreased intracellular Fe2+ levels, thus suppressing erastin- and RSL3-induced ferroptosis. IRP, iron regulatory protein; TFRC, transferrin receptor; FPN, ferroportin; FTH1, ferritin heavy chain 1; ROS, reactive oxidant species; FTL, ferritin light chain.