Fig. 5.

MTA induced Atf6 expression but did not BMP-2 and 4 in MC3T3-E1 cells. Total RNA was extracted, reverse-transcribed, and amplified by real-time PCR using specific primer sets for Bmp2 (A), Bmp4 (B), and Atf6 (D). (C) To determine BMP-2 protein secretion, culture medium was replaced with fresh medium at the indicated time points and the culture was continued with or without MTA for 24 h. The CM was analyzed for BMP-2 concentration using a BMP-2 ELISA Kit. (E) The cells were cultured for 12 days in the absence or presence of MTA at indicated concentration. Whole-cell lysates were subjected to immunoblotting with anti-Atf6 and GAPDH antibodies. (F) A quantitative analysis of the intensity of each band was performed by Molecular Imager FX (Bio-Rad). Total Atf6 was calculated as sum of intensities of p90 and p50 bands. Data represent the means ± SEM (n = 3). Statistical significance of differences between MTA treated group and corresponding time-matched vehicle control is indicated by ***P < 0.001. NS, not significant.