Fig. 7.

NOX2-dependent regulation of IKKε expression in MCF-7 cells. Total RNA from MCF-7 or ZR75.1 cells (A) or from 10 distinct primary breast tumors (B) was extracted, subjected to reverse transcription and specific expression of NOX1-5, DUOX1-2 and actin was evaluated by RT-PCR. Specific positive controls (Ctrl⁺) were used for each gene as detailed in the methods section. In (C), control (CTRL), NOX2-, NOX5- or IKKε-specific RNAi were transfected into MCF-7 cells. Efficiency of NOX2 and NOX5 silencing was controlled by RT-qPCR (left panels). IKKε and TBK1 protein expression were analyzed by immunoblot (IB) using specific antibodies. Actin was used as loading control. IKKε levels were quantified by densitometric analysis using the ImageJ software. Quantification data are expressed as mean±SEM from n≥3. A t-test was performed to compare specific RNAi-transfected to CTRL RNAi-transfected cells. (D) Cell viability was monitored at the indicated times post-RNAi transfection and expressed as % of the corresponding siCTRL-transfected condition at each time point.