HRE reporter gene assay

The pGL3-TK plasmid (Promega, Madison, Wisconsin, USA) with a thymidine kinase minimal promoter (TK-MP) and five repeats of HRE from the phosphoglycerate kinase (PGK) gene were used to construct the HRE-reporter plasmid [27]. The results were normalized against Renilla luciferase, which was co-transfected to correct for variations in transfection efficiency. PC3 and LNCaP cells were grown to 80% confluence in 48-well plates and co-transfected with HRE-reporter plasmid, si-control, and si-PKM2 using Lipofectamine 2000, and then incubated under normoxic or hypoxic conditions for 24 h. The firefly luciferase (FLuc) activity in transfected cells was measured using the luciferase assay system (Promega) and a spectrofluorometer (FL-600 BioTek Instruments, Winooski, VT, USA). Fluorescence values are reported as relative light units (RLUs).