Fig. 6. LPA increases SV40 MES13 cell proliferation by activating the Rho GTPase Rac1.

a Western blots showing the levels of the RhoA, Rac1, and Cdc42 proteins in cells treated with 10 μM LPA for 5 min. RhoA, Rac1, and Cdc42 activity assays were performed using a GST-fusion protein derived from p21-activated kinase or rhotekin, which selectively binds to GTP-bound RhoA, Rac1, or Cdc42. GST–rhotekin precipitates were analyzed using western blotting. b The relative levels of active Rac1 were normalized to the levels of total Rac1 and quantified using ImageJ software (n = 3 independent experiments). c, e After the transfection with the control vector or the dominant-negative Rac1 mutant, SV40 MES13 cells were treated with LPA (10 μM) for 15 min (C) or 6 h (E). Western blots show the levels of p-p38, p38, Rac1, Egr1, KLF5, and β-actin proteins. d, f The relative levels of p-p38, Egr1, and KLF5 were normalized to p38 or β-actin and quantified using ImageJ software (n = 3–4 independent experiments). g Cell morphology was examined using light microscopy (original magnification, × 100), and (h) cell proliferation was examined using the CCK-8 assay (n = 3 independent experiments). *p < 0.05, **p < 0.01, ***p < 0.001. Data are presented as means ± SEM