Fig. 3

p65 and pSTAT3 presented a synergistic effect on G6PD transcriptional regulation. a MatInspector software platform showed that the potential NF-κB- and STAT3-binding sites localized in the G6PD promoter region were adjacent to each other. Primers covering each indicated transcriptional factor–binding region were designed. b ChIP assay was performed with anti-p65 or p50/105 antibodies in ACHN, 786-O, and Caki-1 cells, and the eluate was amplified by real-time RT-PCR with primers covering the pSTAT3-binding site. c Similar experiments were repeated with anti-pSTAT3 or STAT3 antibody in ACHN786-O, and Caki-1 cells, and primers covering the NF-κB-binding site was used. d The interaction between pSTAT3 and p65 was determined by Co-IP in ACHN, 786-O, and Caki-1 cells. e–h The luciferase activity of G6PD-luc WT containing the NF-κB and pSTAT3 binding sites (e, f) and G6PD-luc Deletion without both the NF-κB- and pSTAT3-binding sequence (g, h) were analyzed in 293 T cells following treatment with the STAT3 or NF-κB signaling activator (IL-6, 2 ng/mL or TNFα, 50 ng/mL) (e, g), or inhibitor (STATTIC, 3 μM or BAY11-7082, 2.5 μM) (f, h) independently or jointly for 24 h. Data are expressed as mean ± SD from three independent experiments, each performed in triplicate. *P < 0.05, **P < 0.01, ***P < 0.001, ^#P < 0.05, ^##P < 0.01; ns, nonsignificant vs each control. ST, STATTIC; BAY, BAY11-7082