Figure 2.

siIRE1a enhances the BMP2-mediated osteogenesis assayed by ALP and OCL. (a) siRNA against IRE1a mRNA efficiently inhibited expression of endogenous IRE1a in C2C12 cells. C2C12 cells were infected with either siIRE1a adenovirus or control adenovirus (CTR), and total RNA was collected for real-time PCR analysis. Expression of IRE1a was normalized against the GAPDH endogenous control. The normalized values were then calibrated against the control value, here set as 1. *P<0.05. (b and c) Expression of IRE1a and BMP2 in C2C12 and BMSCs infected with indicated adenoviruses. Cell lysates were prepared from C2C12 (b) and BMSCs (c) infected with siIRE1a1, siIRE1a2, and siIRE1a1+siIRE1a2 adenoviruses, as indicated, and detected by western blotting with anti-IRE1a, anti-BMP2, and anti-tubulin (internal control) antibodies. (d) Semi-quantification of protein relative levels of BMP2 and IRE1α in the C2C12 (b) and BMSC(c) infected with various adenoviruses, as indicated in b and c. Levels were normalized against those of tubulin by MJ Opticon Monitor Analysis Software (Bio-Rad); data were expressed as means±S.D. (n=3). Every treatment group compared with control groups, respectively, *P<0.05 or **P<0.01. (e) siIRE1a increases the BMP2-dependent ALP activity. C2C12 cell lines and BMSCs were infected either Ad-GFP (MOI=50, serves as a control) or BMP2 (300 ng/ml) with or without siIRE1a1, siIRE1a2. The cell lysates were used for determining the ALP activity. (f) siIRE1a enhances the BMP2-dependent OCL production. C2C12 cell lines and BMSCs were infected as described in e, and the cell culture media were used for determining the OCL level