Figure 1

The role of NOX4-mediated ROS generation in human bladder cancer cells. (A) RNA was extracted from human urothelial carcinoma cell lines, T24, UMUC6 and KK47, then mRNA levels of NOX 2, 3, 4 and 5 were analyzed by RT-PCR. mRNA expression of actin was used for (B) T24 cells were treated by ROS detecting fluorescence reagents, CM-DCFDA and DHE, and ROS generation was assessed by fluorescence microscopy (left panel). T24 cells were tarnsfected by NOX4 siRNA at 100 nM and control RNA. After 72 h inoculation, cells were treated with CM-DCFDA and DHE, then intracellular ROS levels were analyzed by flowcytometer as described. Solid black indicates negative control (treated with DMSO). (C) T24 and UMUC6 cells were tarnsfected by NOX4 siRNA at 100 nM and control RNA. After 72 h inoculation, MTS assay, flowcytometric analysis using propidium iodide or western blotting using anti-NOX4, p16 antibody was performed. Actin was used for protein or mRNA expression of housekeeping gene throughout the experiments. Each value is the mean ± SE.