Figure 3. FBP1 inhibits HIF activity in the nucleus.

a, HIF reporter activity measured in RCC4 and RCC10 cells transfected with pHRE-luciferase, in the presence of vector, FBP1 cDNA, or two different FBP1 shRNAs. Transfection efficiencies were normalized to co-transfected pRenilla-luciferase. b, 480 ccRCC tumours from the TCGA database were equally divided into two groups (top and bottom 50% FBP1 expression) based on FBP1 expression levels, and their relative HIF activities were quantified and plotted as described in Methods. c, HIF reporter activity in hypoxic RCC4 and A549 cells (0.5% O₂) with or without ectopic FBP1 expression. d, qRT-PCR analysis of HIF target genes in RCC10 cells expressing vector or FBP1. e, ChIP assays evaluating the chromatin binding of FBP1 to HREs in the GLUT1 promoter, or to a non-hypoxia responsive region of the RPL13A locus. RNA Polymerase II antibody was used as a positive control. f, Immunofluorescent staining of primary human kidney tissue (tubular region) with FBP1 antibody. Arrows point to three representative sites with nuclear FBP1. Rabbit IgG was used as a negative control, and DAPI is a fluorescent nuclear dye. g, Growth of RCC10 cells expressing vector, FBP1, or FBP1 NES (FBP1 linked to a C-terminal nuclear export sequence) cultured in 1% serum. Error bars represent s.d. (three experimental replicates) except in (e), which indicates s.e.m. (three technical replicates from a representative experiment). *p<0.05.