Figure 3. PGJ₂ enhances the cellular IRE activity.

(a) Effect of PGJ₂ on HIF2a translation is mediated through the 5′-UTR HIF2a IRE. Polyclonal 786-O cells stably expressing a luciferase reporter driven by the HIF2a promoter alone or in tandem with HIF2a 5′-UTR deletion mutants were treated with PGJ₂. Mutants are shown on the left as horizontal bars spanning the corresponding UTR area, double vertical lines indicate an inactive IRE mutant. Luciferase activity is reported as a ratio of PGJ₂ treated divided by DMSO treated value, normalized to the reporter with no 5′-UTR. (b) Inhibition of IRP1 is sufficient to block the effect of PGJ₂ on HIF2a activity. 786-O cells were infected with shRNA targeting solely IRP1 (shIRP1), IRP2 (shIRP2) or with shRNAs targeting both IRP paralogs (shIRP1/2). Down regulation of IRP1 and/or IRP2 were confirmed with qRT-PCR and with Western blot (data not shown). These cells were treated with PGJ₂ and EGLN3 mRNA expression was measured by qRT-PCR, normalized to beta-2-microglobulin (B2M). All measurements were done in triplicate and error bars represent standard error of the mean (SEM).