Figure 1. Activated Kras or silenced p16 increased NOX4/p22^phox expression and elevated NOX activity.

(a) The expression of Kras and p16 was analysed by immunoblotting in HPNE control, HPNE/Kras and HPNE/Kras^G12V/shp16 cells. The copy numbers of Kras^WT and Kras^G12V were analysed by qPCR assay in these cells. (b) Heat map of gene expression between HPNE/Kras and HPNE/Kras^G12V/shp16 cells identified using cDNA microoarray. (c) Functional categorization of 614 genes upregulated in response to p16 suppression in HPNE/Kras^G12V cells. (d,f) The expression of NOX4 and p22^phox was analysed by qPCR and immunoblotting in indicated cells. (h) The expression of NOX4, p22^phox and p16 was analysed by immunoblotting in HPNE/Kras^G12V, Colo357 and Capan-2 cells transfected with two independent p16 siRNAs. (e,g,i) NOX activity was determined in indicated cells by measuring NADPH-dependent superoxide (O₂⁻) generation with the lucigenin-enhanced chemiluminescence assay. β-actin was used as the internal loading control. Each bar represents the mean±s.d. Data in d,e,g,i are presented as mean±s.d. (n=3). **P<0.01 for indicated comparison (one-way analysis of variance (ANOVA) with the Newman Keul's multiple comparison test).