Fig. 7.

Silencing of NOX1 inhibited EGF-induced cell invasion, MMP-2 and MMP-9 overexpression, signaling pathways downstream the EGFR in Caco-2 cells, and cell invasion. Caco-2 cells were transfected with or without scramble (si-scramble) or NOX1 (si-NOX1) silence RNAs, for 48 h, and subsequently incubated with or without EGF (10 ng/ml) for A-D, 6 h, E− 10 min and F-24 h. A- NOX1 mRNA levels and B- NOX1 protein levels were evaluated by PCR and Western blot, respectively. C- MMP-2 and D- MMP-9 mRNA levels were measured by qPCR and referred to actin mRNA levels as housekeeping gene. E− Phosphorylation levels of EGFR (Tyr1068), IKK (Ser176/180), p65 (Ser536), Akt (Ser473) and ERK1/2 (Thr202/Tyr204) were evaluated by Western blot. Bands were quantified and values for phosphorylated proteins were referred to the respective total protein content. (F) Caco-2 cells were transfected with si-scramble or si-NOX1 RNAs, and 48 h later replated into Matrigel chambers. Cells were then stimulated with EGF (10 ng/ml) for 24 h, and cell invasiveness was evaluated (20X magnification). Results are shown as means ± SEM of 3–5 independent experiments. Values having different superscripts are significantly different (p < 0.05, One-way ANOVA-test).