Fig. 4

Functional assays for synergistic induction of NF-κB-mediated cytokine expressions. Four HeLa cell-line derivatives with relatively high or low TNFAIP3 and high or low UBE2L3 levels were treated with TNFα for varying periods. Cellular levels of CCL2, CXCL8 (IL8), and IL6 mRNAs were separately measured using qPCR as normalized against GAPDH mRNA. The mRNA levels were measured in triplicate samples and each sample by triplicate qPCR experiments. The nine measurements were averaged per cytokine at each time point. P values for comparisons of cytokine expressions between the reference cell line, transfected with siUBE and siCon, and each of the other three derivatives were calculated in unpaired t tests, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. The synergy (ŝ) is calculated as ŝ = combined effect ÷ (TNFAIP3 reduction effect × UBE2L3 augment effect)