Figure 4. Insulin-promoted osteoblastic differentiation of calcifying vascular smooth muscle cells (CVSMCs) through the ERK1/2/RANKL signaling pathway. (A) Treatment with siRNA- RANKL blocked the expression of RANKL protein in osteoblasts. Cells were treated with siRNA control, or siRNA-RANKL. Total cellular protein was subjected to immunoblot analysis using anti-RANKL antibody. Lane 1, lysate from CVSMCs; lane 2, lysate from CVSMCs treated with siRNA control; lane 3, lysate from CVSMCs treated with siRNA-RANKL. (B) The Insulin Induces Calcification of VSMC via RANKL cells were homogenized for ALP activity assay. Cells were incubated with PD98059 (10 mM), HIMO (10 mM) or LY294002 (10 mM) for 2 h before being treated with 10 nM insulin for 48 h. Cells were also treated with siRNA control or siRNA-RANKL in the presence of 10 nM insulin. (C) Effect of recombinant RANKL protein (15 pM) on the ALP activities decreased by RANKL-siRNA in 10 nM insulin-stimulated CVSMCs after 48 h. (n = 5; **p,0.01 vs. insulin-treated control). doi:10.1371/journal.pone.0029037.g004 antibody a-actin, and by their ability to express high levels of ALP and to form calcified nodules. This investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). The approval was granted by the Second Xiangya Hospital of Central South University Ethics Review Board, China (No. 2010-S163). ALP activity, osteocalcin secretion assay in cultured CVSMCs The cells were washed with PBS, and then the cells layers were scraped into solution. The lysates were homogenized. Then, ALP activity was assayed by spectrophotometric measurement of p- nitrophenol release at 37uC. ALP activity was normalized to total protein content of the cell layer. Osteocalcin released into the culture media was measured using a specific radioimmunoassay kit (DiaSorin Corp., Stillwater, MN, USA). To normalize protein expression to total cellular protein, a fraction of the lysate solution was used in a Bradford protein assay. Real-time quantitative PCR assay for RANKL mRNA expression CVSMCs were plated in 25-cm2 flasks in DMEM containing 10% FBS. After 4 days of culture, cells were subsequently treated with vehicle (serum-free DMEM) or insulin at 1, 5, 10, or 100 nM for 48 h in serum-free DMEM. Cultures were also exposed to fresh serum-free medium with or without 10 nM insulin for 6–48 h. SYBR green real time PCR assay was performed by Light- Cycler technology (Roche) in 20 ml PCR mixture volume consisting of 10 ml of 26 Quantitect SYBR Green PCR Master Mix (Qiagen), containing HotStarTaq DNA polymerase, 500 nMof each oligonucleotide primer and 5 ml of retrotran- scribed cDNA. Preliminary experiments were carried out for primer concentration optimization. Primer sequences for RANKL were 59-CATCGGGTTCCCATAAAG -39 and 59- GAAG- CAAATGTTGGCGTA -39. For GAPDH, the PCR primers were 59-ATCACTGCCACTCAGAAGAC-39 and 59-ACAT- TGGGGGTAGGAACAC-39. Amplifications were performed, and calibration curves were run in parallel in triplicates for each analysis. Each sample was analyzed six times during each experiment. The experiments were carried out at least twice. Amplification data were analyzed using the Sequence Detector System Software (PE Applied Biosystems). Relative quantification were calculated by normalizing the test crossing thresholds (Ct) with the GAPDH amplified control Ct. The results were normalized to GAPDH and expressed as percentage of controls. Soluble RANKL protein secretion assay Levels of RANKL were determined in culture media superna- tants of control cells and cells treated with insulin with commercially available ELISAs (Biomedica, Vienna, Austria). Detection of ERK 1/2 and Akt activation in CVSMCs For studying the effects of insulin on the ERK 1/2 and Akt signaling pathway, Western blot was performed using anti-p-ERK PLoS ONE | www.plosone.org 6 December 2011 | Volume 6 | Issue 12 | e29037