Fig. 6

circPRDM4 recruits HIF-1α onto CD274 promoter to facilitate HIF-1α-mediated transactivation of PD-L1. A Mass spectrum results indicating HIF-1α as a potential binding protein of circPRDM4 under hypoxia. B circRNA pull-down and western blotting assays confirming the interaction between circPRDM4 and HIF-1α under hypoxia. C Colocalization between circPRDM4 and HIF-1α under hypoxia. The profiles of colocalization are demonstrated. D RIP assays showing the binding between circPRDM4 and HIF-1α under hypoxia. E Secondary structure of circPRDM4 was established using RNAfold. F RNA pull-down assays were performed to show that the P1 fragment of circPRDM4 interacted with HIF-1α under hypoxia. G Schematic representation of HIF-1α functional domains and Flag-tagged HIF-1α variants are shown. H The binding between circPRDM4 and the bHLH domain of HIF-1α was examined after transfection of the Flag-tagged HIF-1α variants by RIP using anti-Flag antibody. IgG was used as a control. I Effects of circPRDM4 knockdown on HIF-1α protein expression levels under hypoxia. J Effects of circPRDM4 overexpression on HIF-1α protein expression levels under hypoxia. K ChIP assays were performed to confirm that HIF-1α could bind to CD274 promoter (-500–0 bp). L ChIP assays showed the binding site of HIF-1α on CD274 promoter (from -21 to -12 bp). M The binding between HIF-1α and CD274 promoter was evaluated in circPRDM4-silenced cells using ChIP assays. N Dual-luciferase reporter assays showing that HIF-1α overexpression increased luciferase activities, while circPRDM4 overexpression alone exerted no significant effects on CD274 transcription. Data are shown as mean ± SEM. **, P < 0.01; ***, P < 0.001; ns, no significance