Figure 5

βPix deficiency attenuates MR agonist-induced changes in colon cancer cell function. (a) Extracts from control HCT116 cells and cells transfected with siRNA directed against ARHGEF7 or control siRNA were immunoblotted for βPix. βPix expression was reduced by siRNA directed against ARHGEF7 but not altered by transfection with control siRNA. β-actin was a loading control. (b) βPix contributes to MR agonist-induced colon cancer cell proliferation. Control HCT116 cells and cells transfected with 50 nM siRNA directed against ARHGEF7 or 50 nM control siRNA were stimulated with 100 μM ACh for 48 h. Cell proliferation was measured using the CellTiter-Glo assay. Results are means of at least three independent experiments in triplicate. *, p < 0.05. (c) Extracts from control HT-29 cells and HT-29 cells transfected with siRNA directed against ARHGEF7 or control siRNA were immunoblotted for βPix. βPix expression was reduced by ARHGEF7 siRNA but not altered by treatment with 100 μM acetylcholine (ACh) for 24 h or transfection with control siRNA. β-actin was a loading control. (d) βPix knockdown attenuates colon cancer cell migration. HT-29 cells, placed in the upper chamber of uncoated inserts, were stimulated with 100 μM ACh for 24 h. Cells migrating to the underside of inserts were fixed, stained, and counted using light microscopy. Bars represent mean ± SE from three individual experiments. Symbols represent results from individual experiments (n = 3 per condition). (e) Immunoblotting confirms βPix protein expression was reduced by transfecting HT-29 cells with shRNA directed against ARHGEF7. GAPDH was a loading control. (f) βPix knockdown attenuates ACh-induced human colon cancer cell invasion. HT-29 cells were placed in the upper chamber of Matrigel-coated inserts and stimulated with vehicle or 100 μM ACh for 48 h. Cells invading to the underside of inserts were fixed, stained, and counted using light microscopy. (g) βPix knockdown attenuates ACh-induced colon cancer cell invasion. Bars represent mean ± SE from four individual experiments. Symbols represent individual experiments (n = 4 per condition). (h) shRNA knockdown of ARHGEF7 expression reduced PTGS2 mRNA levels p < 0.001). mRNA levels were measured by qPCR and normalized to β-2 macroglobulin. MW, molecular weight. Original uncut immunoblots for (a, c), and e are shown in Supplemental Materials.