Fig. 4: IL-17-induced STEAP4-mediated copper uptake enhances the E3-ligase activity of XIAP.

a IL-17 stimulation accentuates copper-induced XIAP shift. LS174T cells were pretreated with 10 µM Cu(II) for 24 h and then IL-17 was treated at the indicated time points. b STEAP4 overexpression accentuates XIAP shift. STEAP4 was induced by 1 μg/mL doxycycline for 24 h in the inducible clone and then treated with indicated concentrations of Cu (II) for 16 h. c STEAP4 is required for IL-17-induced accentuation of XIAP shift. Wild-type (WT) and STEAP4 knockout (St4 KO) intestinal organoids were first exposed to 10 μM Cu(II) for 16 h and then treated with IL-17 for indicated time points. d Western blots analysis for XIAP sustaining NF-κ signaling. XIAP WT and KO cells were pretreated with or without 10 μM copper, followed by IL-17 treatment for the indicated time points. e XIAP inhibits Caspase-3 cleavage. XIAP WT and KO cell line were first primed with IL-17 for 12 h and then treated with 5-FU for the indicated time points. f In vitro ubiquitination assay using recombinant XIAP prepared in the presence (XIAP-Cu) or absence (XIAP) of copper and cleaved human caspase-3 as substrate. g In-gel SOD activity assay in wild-type and XIAP knockout Ls174t in response to IL-17 stimulation. h Coimmunoprecipitation analysis of XIAP interaction with indicated protein in response to STEAP4 expression in STEAP4-inducible clone. i Coimmunoprecipitation analysis of endogenous XIAP in response to STEAP4 expression. Data represent three independent experiments with similar results.