Fig. 2

NOX-2 derived ROS enhances MMP-7 expression and invasion of TPA-treated colon cancer cells. a Superoxide production in TPA (12 ng/ml)-treated HT29 and SW620 cells was measured by chemiluminescence (CL) using lucigenin (400 μM). b HT29 cells were pretreated with Apo (100 μM), DPI (10 μM), Cele (20 μM), ANT (10 μM), or Vit. E (50 μg/mL) for 1 h prior to TPA treatment for 1 h. Intracellular ROS level using DCF-DA (upper panel) and invasion (lower panel) were determined by fluorescence microscopy and Matrigel invasion assay, respectively. The bar graph represents quantitative data on ROS as measured by lucigenin chemiluminescence assay and invasion. Apo, apocynin; DPI, diphenyleneiodonium; Cele, celecoxib; ANT, antimycin A. *P < 0.05 compared to control group. ^# P < 0.05 compared to TPA-treated cells. c Basal mRNA expression of NADPH oxidase components was measured by qRT-PCR in HT-29 and SW620 cells. The data represents mean ± SEM from three independent experiments. *P < 0.05 compared to HT29 cells. d Basal superoxide production normalized by cellular protein content was compared between HT29 and SW620 cells. *P < 0.05 compared to HT29 cells. e-h Cells were transfected with siRNA specific to NT, NOX1, NOX2, or p67phox. Basal NADPH oxidase activity (e) was measured by using lucigenin chemiluminescence. *P < 0.05 compared to NT-treated group. TPA-induced expression of NOX1, NOX2, and MMP-7 (f), production of ROS (g, upper panel of each cell line), and invasion (g, lower panel of each cell line) were examined in both HT29 and SW620 cells. The bar graph represents the NADPH oxidase activity measured by lucigenin chemiluminescence using protein extracts after transfection with siRNA (h) and number of invaded cells (i). *P < 0.05 compared to vehicle-treated control group. ^# P < 0.05 compared to TPA-treated cells