Angiotensin II Activates the Calcineurin/NFAT Signaling Pathway and Induces Cyclooxygenase-2 Expression in Rat Endometrial Stromal Cells
Figure 5
Inhibition of endogenous CN-NFAT binding with the exogenously expressed VIVIT peptide blocked COX-2 protein expression.
(A and B) Primary stromal cells isolated from uteri of non-pregnant rats were transfected with 800 ng of expression constructs encoding either pEGFP-VIVIT or pEGFP-N1 as control. Transfected cells were stimulated for 14 h with PIo, or left untreated (ns). Cells were permeabilized and stained for COX-2. Detection of cells co-expressing both COX-2 and GFP protein was analyzed by two color-FACScan. (A) Density plots showing FACScan analysis of transfected cells immunostained for COX-2 protein. The percentage of acquired cells in each quadrant is embedded. (B) Upper panels: Histograms showing COX-2 immunostaining of gated, GFP positive- cells. Left: pGFP-N1- transfected cells; rigth: pGFP-VIVIT- transfected cells. Closed gray histogram: non-stimulated (ns) cells; open black histogram: (PIo)- stimulated cells. The percentage of COX-2-positive cells (% positive) and the mean fluorescence intensity (MFI) from each histogram are enclosed. Lower panel: Bar graphs showing the product of the percentage of COX-2-positive cells and the mean fluorescence intensity, from GFP-positive cells ploted in upper panels. Open bars: cells transfected with pEGFP-N1; dark bars: cells transfected with the pEGFP-VIVIT. (A–B) The results shown are from a representative experiment of three performed, and values are the means ±SD of duplicate determinations. *** P<0.001; ** P<0.01 (ANOVA).
