Figure 4.

Silencing of ISD11 diminishes mitochondria and cytosolic aconitase activities and activates iron-responsive element (IRE)-binding activity. (A) Aconitase activity assay revealed reduced activity of Fe–S cluster enzymes in cells treated with ISD siRNA [WT, wild-type; (−)Ctrl, negative control; Mock, treated with Lipofectamine 2000 reagent; oligo, siRNA of ISD11]. (B) Western blots of cells transfected with ISD11 siRNA revealed that (iron-regulatory protein) IRP1 and α-tubulin levels (loading control) did not significantly change. Results shown are representative of four independent experiments. (C) Xanthine oxidase activity assay shows obvious decrease in the RNAi-treated cells compared with the untreated wild-type cells. (D) Lactate dehydrogenase activity assay shows no change in the RNAi-treated cells compared with the untreated wild-type cells. (E) Gel retardation assays of IRPs. Transfected cells were harvested nine days after three times successive transfection and analyzed for total binding activity of IRP1 and IRP2 to ³²P-labeled IRE of human ferritin mRNA. Lysates of WT and ISD11 knock-down HeLa cells (10 µg protein/lane) were incubated with ³²P-IRE and resolved on a 10% non-denaturing gel. Binding of IREs to IRPs were visualized by autoradiography in the absence of β-mercaptoethanol and quantified using a phosphorimager. Both IRPs have increased IRE-binding activity. (WT, wild type; (−)Ctrl, negative control; oligo2, siRNA of ISD11). IRP1- (bottom band) and IRP2 (up band)-IRE complex. (F) Quantitative analysis of IRP2 levels by western blot revealed that IRP2 levels increase in ISD11-depleted cells (top panel) and the result is reproducible (bottom panel).