Fig 2: HIF-1a modulates PRKAR2B expression via Sp1 promoter binding and their expression levels are inverse correlated in acromegalic tumors.a, d Chromatin immunoprecipitation showing decreased Sp1 binding on the Prkar2b promoter in GH3 cells overexpressing HIF-1a. No DNA binding was quantified with HIF-1a. Rabbit IgG was used as control. b, e Co-immunoprecipitation experiment showing that both HIF-1a and HIF-1aR30A physically associate with Sp1. HIF-1a and Sp1 immunoprecipitates were blotted for Sp1. Proteins immunoprecipitated with rabbit IgG were used as controls. c, a Expression of PRKAR2B gene in normal pituitary glands (n = 9) and acromegalic tumors (ACRO, n = 10). Data are PRAKR2B/TFIIB. *P = 0.006 (U-test). d, b Western blot analysis of normal pituitary (NP) and GH-secreting tumors (ACRO) showed a significant loss of PRKAR2B protein expression, whereas HIF-1a protein levels measured in the same tumors are significantly increased in ACRO vs NP (n = 4 per group due to scarcity of material for dual measurement). e, c Linear regression analysis of PRKAR2B transcript and HIF-1a protein levels in 14 acromegalic tumors. PRKAR2B was determined by real-time RT-PCR and values are PRKAR2B/TFIIB arbitrary units. HIF-1a protein was quantified from immunoblots (Chemidoc). Kendall’s t = -0.663, rt = -0.480, P = 0.009.

Fig 3: Effects of functional HIF1alpha suppression on target gene expression in cumulus cells derived from COCs as evaluated by semi-quantitative (TaqMan) real time PCR. Three experimental groups were involved: immature COCs (Immat.), control in vitro matured COCs (IVM/control), and COCs treated with echinomycin (5 nM) during IVM (IVM/E). The effect of functional HIF1alpha suppression on gene expression of target genes in cumulus cells was tested by one-way ANOVA, revealing: HAS2 (P < 0.0001), TNFAIP6 (P < 0.0001), TMSB4 (P = 0.0001), TMSB10 (P = 0.005), GATM (P < 0.0001), NPR2 (P = 0.036), GLUT1 (P = 0.0008), CX43 (P = 0.0001), COX2 (P = 0.001), PTGES (P < 0.0001), AKR1B1 (PGFS/20aHSD) (P < 0.0001), STAR (P = 0.0001), and HSD3B (P < 0.0001). Tukey–Kramer multiple comparisons post-test was applied to test the effect of treatment on gene expression (solid lines). Bars with asterisks differ at: *P < 0.05, **P < 0.01, ***P < 0.001, as indicated on the figure. Results are presented as mean ± SEM.

Fig 4: Effects of the suppression of HIF1alpha expression by PX-478 on target gene expression in cumulus cells derived from COCs as evaluated by semi-quantitative (TaqMan) real time PCR. The following experimental groups were involved: immature COCs (Immat.), control in vitro matured COCs (IVM/control), and COCs treated with two dosages of PX-478 (25 and 50 µM) during IVM (IVM/25 µM PX-478 and IVM/50 µM PX-478). The one-way ANOVA was applied to test the effect of HIF1alpha suppression on gene expression in treated cells, revealing: HIF1A (P = 0.0016), HAS2 (P = 0.018), TNFAIP6 (P = 0.0006), TMSB4 (P < 0.0001), TMSB10 (P = 0.0002), GATM (P = 0.002), NPR2 (P < 0.0001), GLUT1 (P = 0.004), CX43 (P = 0.06), COX2 (P = 0.001), PTGES (P = 0.005), AKR1B1 (P = 0.004), STAR (P = 0.002), and HSD3B (P = 0.002). Additionally, the HIF1A protein levels were determined by Western blot analysis in all experimental groups, i.e., immature, as well as in control and PX-478 (50 µM) and echinomycin (5 nM) treated IVM groups. The one-way ANOVA was applied revealing P < 0.0001. Tukey–Kramer multiple comparisons post-test was applied to test the effect of treatment on gene and/or protein expression (solid lines). Bars with asterisks differ at: *P < 0.05, **P < 0.01, ***P < 0.001, as indicated on the figure. Results are presented as mean ± SEM.

Fig 5: BAF180 is required for strong expression of HIF1 and HIF2 target genes in hypoxic (HX) Hep3B cells and pVHL-deficient ccRCC cell lines. (a) Western blot analysis of BAF180, HIF1a and HIF2a proteins in normoxic (NX) and HX Hep3B cells stably transduced with non-targeting control (SCR shRNA) or BAF180 shRNA. (b–e) qRT–PCR analysis of messenger RNA (mRNA) levels of HIF1 target genes (b), HIF2 target genes (c), HIF1/HIF2 common target genes (d) and BRG1-independent HIF target genes (e) in NX and HX Hep3B cells stably targeted with control or BAF180 shRNA. (f) qRT–PCR analysis of mRNA levels of known HIF2 target genes in 786-O/Tet-on Cas9 cells with no sgRNA or BAF180 sgRNA #1 (right). Expression levels of BAF180, HIF2a and ARNT protein in these cells were also shown (Left). (g) qRT–PCR analysis of mRNA levels of known HIF1 and HIF2 target genes, as well as HIF and ARNT in RCC4 cells stably expressing GFP (control) or BAF180 protein. Clones #12 and #14 express low and high BAF180 protein levels, respectively.