Fig. 1. Shear flow activates p38, Rac1 and NFB in colon cancer cells. (A) SW480 cells were exposed to shear stress (15 dyn/cm²) for 0 to 60 minutes. MAPK pathway activity was measured by western blot of phospho-ERK, -JNK and -p38. (B) HT29 cells exposed to shear stress (15 dyn/cm²) for 0 to 60 minutes. (C) Rac1 activity in SW480 cells exposed to shear stress. GTPS treatment increased the sensitivity of the GST-PBD pull-down assay. SW480 cells were exposed to shear stress (15 dyn/cm²) for 0, 2, 5 and 20 minutes. Lysates were treated with GTPS and GST-PBD pull-down was performed, followed by Rac1 western blot. (D) SW480 cells were exposed to shear stress for 0 to 12 hours and lysates were collected. Total RNA was purified and DKK1 mRNA concentration was analyzed by real-time qPCR. Cells were treated with the JNK inhibitor SB600125 and the p38 inhibitor SP202190 for 1 hour prior to initiation of shear. Cells were collected and DKK1 mRNA was analyzed by real-time qPCR. Inset in D, western blot analysis of DKK1 expression in SW480 cells exposed to shear stress for 0 to 12 hours. (E) As D, using HT29 cells. All experiments were performed in triplicate with consistent and repeatable results. (F) SW480 cells were transiently transfected with 1 µg/ml of NFB reporter and either 1 µg/ml of DN IKK or DN IKK and exposed to 15 dyn/cm² of shear stress for 12 hours. Asterisk indicates significant difference in comparison with control (Student's t-test, P<0.01).