Figure 3. RelA NF-kB transcription factor is required for cytokine gene activation in ARID1A/PIK3CA mutant cells.

(A) The NF-kB inhibitor IKK-IIV, but not a STAT inhibitor Ruxotinilib, inhibited cytokine expression in the T80 (AM) cells. T80 (AM) cells were treated with DMSO, Ruxotinilib (1μM) or IKK-IIV (1μM) for three days. Control cells were treated with DMSO. Expression of selected cytokines genes were measured by qRT-PCR, normalized by β-actin (n=3). Error bars, S.E.M.

(B) RelA knock-down reduced cytokine gene expression. Control and T80 (AM) cells were transfected as indicated and the expression of selected cytokines genes were analyzed by qRT-PCR three days after transfection (n=4). Error bars, S.E.M.

(C) RelA inhibition by a dominant-negative IkB partly reduced cytokine gene expression. T80 (AM) cells were infected with empty or IkBα-SR expression retrovirus vector. After selecting with 100 ng/ml Zeocin for two days, surviving cells were analyzed by qRT-PCR (n=4). Error bars, S.E.M.

(D) Cytokine genes are direct RelA targets. ChIP analysis was performed in the indicated cells using antibodies against rabbit IgG (control) or RelA. Immunoprecipitated samples were PCR amplified using primers that amplify the RelA elements of the indicated genes. The signals were normalized with input (n=3). IgG of control cells was set as 1. Error bars, S.E.M. Statistical analysis comparing RelA ChIP in AM and control cells is presented.

*, p-value < 0.05; **, p-value < 0.01; ***, p-value < 0.001. See also Figure S3.