Angiotensin II Activates the Calcineurin/NFAT Signaling Pathway and Induces Cyclooxygenase-2 Expression in Rat Endometrial Stromal Cells
Figure 3
NFAT dephosphorylation and nuclear localization was abrogated by CN inhibition in primary rat ESC.
(A) Immunoblots of whole extracts of primary cultured ESC isolated from uteri of non-pregnant rats showing endogenous expression of NFATc1 and β-actin as loading control. Primary cultures of cells isolated from uteri of non-pregnant rats were pretreated for 1 h with vehicle (lanes 1,3, 5) or 200 ng/ml of CsA (2, 4, 6), and then exposed for 2 h to PIo (lanes 3 and 4); 500 nM of Ang II (lanes 5 and 6), or were left unstimulated (ns, lanes 1 and 2). The position of phosphorylated NFAT (P-NFATc1) and dephosphorylated NFATc1 (NFATc1) is indicated. Right panel bar plot shows the densitometric data analysis of the results shown in the left panel. The NFAT/β-actin ratio was calculated and then the NFATc1/(NFATc1+NFATc1-P) ratio was plotted. The values are the means ± SD obtained from three independent experiments performed. Open bars, cells pre-treated with vehicle; closed black bars, cells pre-treated with CsA ** P<0.01; * P<0.05 (ANOVA). (B) Immunofluorescence analysis of endogenous NFAT protein with anti-NFATc1 antibody (c–l) or nonimmune Ab (a and b) as control of unspecific staining. Primary cultures of cells isolated from non-pregnant rats were pretreated for 1 h as before with vehicle (c–h) or CsA (i–l), and then exposed 2 h to PIo (e–f, i–j); 500 nM of Ang II (Ang, g–h, k–l), or were left unstimulated (ns, c–d). (a, c, e, g, i, k): FITC staining of the cells. (b, d, f, h, j, l): nuclei staining with DAPI. Magnification 200×. Shown is a representative experiment out of three performed.
