Fig. 3. Effects of BD on the expression of some crucial genes and proteins in osteogenic-induced MC3T3-E1 cells. The qRT-PCR (a, b) and WB (c, d) analyses were performed to examine the expression of Bmp2, Ocn, Runx2, Opg, Rankl genes and partial relative proteins in MC3T3-E1 cells after the addition of BD to the osteogenic medium and subsequent 7-d incubation. A further WB assay (e, f) was performed to examine the level of Smad5 phosphorylation （P-Smad5） in MC3T3-E1 cells following BD or LDN-193189 (LDN) addition to the osteogenic medium and subsequent 7-d incubation. (a) BD increased the expression of Bmp2, Ocn, and Runx2. (b) BD increased the expression of Opg and the ratio of Opg/Rankl, while it decreased the expression of Rankl with the most pronounced effect observed at 1.00 μmol/L. (c, d) BD increased the expression of OPG, while it decreased the expression of RANKL, especially at 1.00 μmol/L. (e, f) BD increased the level of Smad5 phosphorylation, while this promoting effect disappeared with the simultaneous use of LDN-193189. Data are expressed as mean±SD (n=3). ^* P<0.05, ^** P<0.01, ^*** P<0.001, and ^**** P<0.0001 represent comparisons with the control group; ^# P<0.05, ^## P<0.01, and ^### P<0.001 represent comparisons between experimental groups. BD: benidipine; qRT-PCR: qualitative reverse transcription polymerase chain reaction; WB: western blot; LDN-193189: a selective inhibitor of bone morphogenetic protein type Ⅰ receptor; Bmp2: bone morphogenetic protein 2; Ocn: osteocalcin; Runx2: Runt-related transcription factor 2; Opg: osteo-protegerin; Rankl: receptor activator of nuclear factor-κB ligand.