Figure 2. IRP2 binding to IRE is inhibited by cisplatin.

A) Binding of IRPs to IRE was assessed by pull-down assays using whole cell lysates from SW480 cells incubated with cisplatin (0, 5, 10, 25 µg/mL) for 24 h, DFO (100 µM) or FAC (250 µM) for 10 h. Whole cell lysates were incubated with biotinylated ferritin H IRE probe (Biotin-FH IRE) and precipitated with streptavidin beads, followed by Western blotting with IRP1 and IRP2 specific antibodies. Coomassie Brilliant Blue (CBB) staining for verification of equal protein loading. IRP1, IRP2, and GAPDH protein levels in the whole cell lysates were measured by Western blotting (WB). B) Binding of IRPs to ferritin IRE was assessed by RIP assays. SW480 cells stably transfected with pcDNA3.1/C-HA (Empty), HA-IRP1, or HA-IRP2 plasmid were treated with 25 µg/mL cisplatin for 22 h. Whole cell lysates were immunoprecipitated with anti-HA antibody (IP) and 10% of IP samples were subjected to Western blot with anti-HA antibody to check IP efficiency (top Western blots). IgG signal as a loading control. The same IP samples were used for RIP assays for detection of ferritin H (FH) or β2-microglobulin (B2M, negative control) mRNA enrichment by qPCR (bottom graph). mRNA enrichment was normalized by input total RNA, and the results were shown as relative mRNA enrichment by IRP2 (- cisplatin, 100%). Mean ± SD (n = 3). C) siRNA for control (siCtrl), IRP1, or/and IRP2 was transfected into SW480 cells and whole cell lysates were used for Western blots with IRP1, IRP2, TfR1, ferritin H (FH), ferritin L (FL) or GAPDH antibodies. D) Whole cell lysates were incubated with 10 µg/mL cisplatin for 0, 3, 6, 18, 24 h, 25 µg/mL carboplatin for 24 h, 100 µg/mL DFO for 6 h, or 250 µg/mL FAC for 6 h in test tubes, and pull-down assay was performed using a biotin-FH IRE probe for detection of IRE-bound IRP2 by Western blotting. * indicates a non-specific band. CBB staining is shown for verification of equal loading. E) Human Myc-Flag-tagged IRP2 protein was incubated with cisplatin or carboplatin in the presence of ³²P-labeled ferritin H IRE probe. IRP2-IRE complex was separated from the free IRE probe by electrophoresis and visualized by autoradiography. Control IgG or anti-Flag antibody was incubated with samples of Myc-Flag-IRP2 mixed with ³²P-IRE probe for verification of the IRP2-IRE interaction as a super shift (left two lanes). F) Human Myc-Flag-tagged IRP2 protein was incubated with indicated concentrations of cisplain, carboplatin (top), or cisplatin (200 µg/mL), H₂O₂ (0.1 mM, 1 mM) or tert-butylhydroperoxide (tBHP, 0.1 mM, 1 mM) (bottom) overnight at room temperature. Samples were loaded on native polyacrylamide gel electrophoresis and subjected to Western Blotting with anti-IRP2 antibody. G) A biotin-FH IRE probe mixed with streptavidin beads was pre-incubated with cisplatin, carboplatin or PtCl₂ overnight. Then, the complex was washed to remove unbound platinum compounds and re-incubated with SW480 whole cell lysates, and pull down assay was performed for detection of IRP2-IRE interaction by Western blotting. CBB staining for verification of equal protein loading.