Fig 1: 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.
Fig 2: Knockdown of IRP1 and IRP2 inhibits erastin- and RSL3-induced ferroptosis. (A) IRP1 and IRP2 knockdown inhibited erastin- and RSL3-induced ferroptotic cell death in A375 melanoma cells. (B) Knockdown of IRP1 and IRP2 inhibited erastin- and RSL3-induced iron accumulation in A375 cells. (C and D) Knockdown of IRP1 and IRP2 suppressed erastin- and RSL3-induced (C) MDA and (D) lipid ROS accumulation. The black line represents the control cells; the red line represents IRP1 knockdown; the green line represents IRP2knockdown and the blue line represents IRP1 and IRP2 knockdown. (E) Protein levels of IRP1 and IRP2 were detected following IRP1 and IRP2 knockdown. Data are presented as the mean ± SD of three independent experiments. *P<0.05; ***P<0.001; ****P<0.0001. IRP, iron regulatory protein. IRP, iron regulatory protein; sh, short hairpin RNA.
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: IRP1 function in ferroptosis depends on TFRC, FPN and FTH1. (A) Overexpression of TFRC and knockdown of FPN and FTH1 enhanced erastin- and RSL3-induced ferroptotic cell death in IRP1 knockdown A375 melanoma cells. (B) Overexpression of TFRC and knockdown of FPN and FTH1 promoted erastin- and RSL3-induced iron accumulation in IRP1 knockdown A375 melanoma cells. (C and D) Overexpression of TFRC and knockdown of FPN and FTH1 promoted erastin- and RSL3-induced (C) MDA accumulation and (D) lipid ROS accumulation in IRP1 knockdown A375 melanoma cells. The black line represents the control cells; the red line represents IRP1 knockdown; the green line represents overexpression of TFRC and IRP1 knockdown; the blue line represents the FPN, FTH1 and IRP1 knockdown and the orange line represents overexpression of TFRC and FPN, FTH1 and IRP1 knockdown. (E) Protein levels of IRP1, TFRC, FPN and FTH1 in A375 cells transfected with the indicated constructs. Data are presented as the mean ± SD of three independent experiments. *P<0.05; **P<0.01; ***P<0.001. IRP, iron regulatory protein; TFRC, transferrin receptor; FPN, ferroportin; FTH1, ferritin heavy chain 1; sh, short hairpin RNA; MDA, malondialdehyde; n.s., not significant.
Fig 5: IRP1 is essential for the expression of iron-regulating proteins in ferroptosis. (A) Knockdown of IRP1 suppressed the mRNA expression levels expression of TFRC in A375 melanoma cells following erastin and RSL3 treatment. (B and C) Knockdown of IRP1 had no significant effect on the mRNA expression levels of (B) FPN and (C) FTH1 following erastin and RSL3 treatment. (D) Protein levels of TFRC, FPN and FTH1 in A375 cells treated with erastin or RSL3. Data are presented as the mean ± SD of three independent experiments. ***P<0.001; ****P<0.0001. IRP, iron regulatory protein; TFRC, transferrin receptor; FPN, ferroportin; FTH1, ferritin heavy chain 1; sh, short hairpin RNA; n.s., not significant.
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