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. 2013 Mar 28;451(Pt 2):185–194. doi: 10.1042/BJ20130026

Figure 1. USP52 is a regulator of the hypoxia response.

Figure 1

(A) U2OS osteosarcoma cells stably expressing a hypoxia reporter construct consisting of three tandem HREs fused to the firefly luciferase gene (U2OS-HRE) were used to identify novel mediators of the hypoxia response. Western blot analysis demonstrates that HIF1A protein expression increases concomitant with a 10-fold increase in luciferase activity upon hypoxia treatment. Tubulin was used as a loading control. (B) Deconvolution analysis of USP52 revealed that individual siRNAs si1 and si3 both elicited an impaired response to hypoxia that correlated with the ability of these oligonucleotides to reduce USP52 protein levels by approximately 50%. Tubulin was used as a loading control. (C) Western blot of U2OS-HRE cells stably expressing siRNA-resistant YFP–USP52 were resistant to the individual siRNA as indicated, but remained sensitive to the other siRNA. (D) Luciferase assays of siRNA-resistant cells lines revealed USP52 si1 treatment was rescued to 80%, whereas si3 treatment was rescued to 95%. Cell lines remained sensitive to the individual siRNA to which resistance was not designed. Molecular masses are indicated in kDa in the blots, and results in histograms are means±S.E.M.