FIG. 3.
Overexpression or silencing of NOX1 modulates the expression of transcription factors involved in cell lineage differentiation. HT-29Cl.16E and Caco-2 cells were transfected with pcDNA3-NOX1 or pEBV-shRNA-NOX1, respectively, or with the corresponding empty vectors. Semiquantitative RT-PCR analysis of NOX1, NOXO1, NOXA1, p22phox, and rac1 mRNA levels (A) or of transcription factors involved in cell fate (C) during cell proliferation (day 3) and differentiation (days 13 to 23) is shown. GAPDH and β-actin mRNA levels were used as controls for HT-29Cl.16E and Caco-2 cells, respectively. The ratio between each mRNA level and that for GAPDH or β-actin was quantified by scanning densitometry. Values are representative of 3 individual experiments performed in triplicate; means ± SEM are shown. *, P < 0.001 relative to empty vector. (B) PMA-induced ROS production in transfected HT-29Cl.16E and Caco-2 cells was measured by luminol-enhanced chemiluminescence in the presence or absence of DPI, an inhibitor of flavoproteins. Values are means ± SEM for three independent experiments. *, P < 0.001 relative to results with empty vector.