Figure 8
ADMA induced myofibroblast activation and its effect on ROS-NOX4-ERK signaling axis in rat mesangial cells: (A) RMC cells pre-treated with ARG/GW4064 for 30 min and then treated with HG/ADMA for 10 h in transwell and the pictures were taken in four random high power fields. Cells were counted from four random microscopic fields per insert in triplicate. The migrated cell numbers were normalized to that of the control group. The results were expressed as % of invasion. (B) RMC monolayers pre-treated with ARG/GW4064 for 30 min were scratched by using pipette and treated with HG/ADMA for 12 h, the percentage of wound width was calculated and the results were expressed as % of migration compared to control. (C) Cell proliferation was determined by BrdU incorporation assay, and the results were expressed as % of proliferation compared with control. (D) Cells pretreated with ARG/GW4064 for 30 min before HG/ADMA treatment and intracellular ROS generation was analysed by multimode reader. The data are represented as % of Relative Fluorescence Unit (RFU). Cells were treated with ADMA in (F,H) time and (E,G) dose dependent manner and cells were harvested after respective conditions. NOX-4, p-ERK and t-ERK of respective conditions were detected by immunoblot with GAPDH as loading control. Results are presented as Mean ± SD of three independent experiments. (A–D) *p < 0.05 compared to the Control, #p < 0.05 compared with HG, $p < 0.05 compared with ADMA. (E,G) *p < 0.05 compared with Control, $p < 0.05 compared with ADMA (10 μM), #p < 0.05 compared with ADMA (20 μM). (F,H) *p < 0.05 compared with Control, #p < 0.05 compared with 30′, $p < 0.05 compared with 1H, &p < 0.05 compared with 3H. Original uncropped images of the blots are given in Supplementary Fig. S11.
