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. 2011 Jun 9;6(6):e20780. doi: 10.1371/journal.pone.0020780

Figure 2. PTH-CREB pathway promotes osteoblast differentiation.

Figure 2

(A) ALP activity in C2C12 cells, treated with PTH at 0 to 200 nM for 24 hours, was measured with normalization by cell proteins. * p<0.05 (vs vehicle; mean±SE, n = 8). (B) mRNA levels of Runx2 and Col1a1 in C2C12 cells, treated with PTH for 12 hours, were quantitated by real time PCR, normalized by 18S rRNA. * p<0.01 (vs vehicle; mean±SE, n = 6). (C) ALP activity in C2C12 and 2T3 cells was measured after transfection with CREB expression plasmid and treated with noggin at 500 nm/ml for 48 hours. * p<0.01 (CREB vs vector); # p<0.05 (noggin vs vehicle; mean±SE, n = 8). (D) mRNA levels of Runx2, Col1a1 and OCN in C2C12 cells, transfected with CREB for 24 hours, were measured by real time PCR, normalized by 18S rRNA. * p<0.05 (vs vector; mean±SE, n = 6). (E) 2T3 cells were transfected with vector (top) or CREB plasmid (bottom) and cultured under osteogenic conditions for 14 and 21 days. Von Kossa staining was performed to visualize the mineralized matrix formation. (F) Isolated calvarial osteoblastic cells from newborn conditional CREB knockout mice (CREB cKO) and their CREBfloxed littermate controls were cultured for 24 hours and ALP activity was measured. * p<0.05 (cKO vs control); # p<0.05 (PTH on cKO vs PTH on control); ** p<0.05 (PTH va vehicle on control mice; mean±SE, n = 6); NS: not significant.