Phospholipase Cε Modulates Rap1 Activity and the Endothelial Barrier
Fig 2
PLCε depletion leads to increased RhoA activity and stress fiber formation.
A) Active RhoA pulldown assay from lysates infected with negative control (NC) or PLCε siRNA ± 1μM 8-pCPT-2′-O-Me-cAMP-AM (cPT-2OMe). Blots are representative, n = 4. B) Densitometric quantification of blots in (A). Data shown are active RhoA normalized to total RhoA ± SEM, n = 4, p = 0.0005 by ANOVA, *p<0.05 vs. NC infected cells, and **p<0.001 vs. PLCε siRNA infected cells by Tukey’s Multiple Comparison Test. All blots used for this quantification can be found in S5 Fig. C) Rhodamine-phalloidin stained HPAEC infected with NC or PLCε siRNA ± siRNA resistant PLCε. Scale bar = 50μm. Images are representative of 3 separate experiments. D) Fluorescent intensity quantification of (C). n≥40 cells from 10 fields of view, p<0.0001 by ANOVA, *p<0.001 by post-hoc test vs. NC infected cells. ** p<0.001 vs. PLCε siRNA infected cells. E) Epifluorescence images of rhodamine-phalloidin stained HPAEC infected with negative control (NC) or PLCε siRNA ± 1μM 8-pCPT-2′-O-Me-cAMP-AM. F) Fluorescent intensity quantification of cytoplasmic stress fibers (E). n≥40 cells from 10 fields of view, p<0.0001 by ANOVA, *p<0.001 by post-hoc test vs. NC infected cells. ** p<0.001 vs. PLCε siRNA infected cells. G) Fluorescent intensity quantification of cortical actin in (E). n≥40 cells from 10 fields of view, p<0.0001 by ANOVA, *p<0.001 by post-hoc test vs. NC infected cells. ** p<0.001 vs. PLCε siRNA infected cells.