Figure 6.

CCNT2 binds VEGFA promoter and enhancer to regulate VEGFA elongation rate and alternative splicing. (A andB) K562 cells were transfected with siRNA against GFP as negative control and siRNA targeting PML, CCNT2 and MAZ for 72 h. RNA was analyzed by real time PCR for VEGFA₁₂₁ and VEGFA₁₈₉ relative to VEGFA total mRNA amount. PSI was calculated by VEGFA₁₈₉ relative to VEGFA₁₂₁ (A); and for nascent pre-mRNA VEGFA relative to VEGFA total mRNA amount (B). (C) Enhancer mutated clones 9 and 26 were transfected with siRNA against GFP as negative control and siRNA targeting PML, CCNT2 and MAZ for 72 h. RNA was analyzed by real time PCR for VEGFA₁₂₁ and VEGFA₁₈₉ relative to VEGFA total mRNA amount. PSI was calculated by VEGFA₁₈₉ relative to VEGFA₁₂₁. (D) Nascent mRNA production in WT K562 cells transfected with siRNA against GFP as negative control and siRNA targeting CCNT2 in different regions of the VEGFA gene after release from DRB-inhibition. Values represent mean ± SD of three independent experiments (* P< 0.05; ***P< 0.001).