FIGURE 3.
NFATc2 suppresses Notch canonical signaling in osteoblasts. RosaNotch calvarial osteoblasts were transduced with Ad-CMV-caNFATc2 (NFATc2, black bars) or control Ad-CMV-GFP (Control, white bars) and co-transduced with Ad-CMV-CRE (CRE) to induce Notch or control Ad-CMV-GFP (GFP). In A, osteoblasts were cultured to subconfluence and transiently transfected with the 12×CSL-Luc reporter or with the Hey1-Luc or Hey2-Luc promoter constructs, co-transfected with a CMV/β-galactosidase expression vector, and harvested after 48 h. Data shown represent luciferase/β-galactosidase activity. Values are means ± S.E., n = 6. In B, osteoblasts were cultured for 72 h after confluence, and total RNA was extracted and amplified by qRT-PCR. Data are expressed as ratio of Hey1 or Hey2 copy number, corrected for Rpl38 expression, relative to corrected expression in control cells co-transduced with Ad-CMV-GFP. Values are means ± S.E., n = 4. *, significantly different between NFATc2 and control, p < 0.05. +, significantly different between CRE and GFP, p < 0.05. Data are from representative experiments performed in duplicate.