Figure 1

Effect of pulse electromagnetic fieldsl (PEMF) on primary cilia A, immunostaining for primary cilia; B, after exposure at the 50 Hz 0.6 mT PEMF for 1.5 h, then put the cells in CO2 cell incubator for 2 h, and the primary cilia get longer; C, cell with primary cilia; D, the length of primary cilia; Con, control; *P<0.05, vs. control."

Figure 2

Treatment with 50 Hz 0.6 mT pulse electromagnetic fields (PEMF) enhances the expression of PI3K The protein level was examined by Western blotting. Prorein in lysates were separated by 12% SDS-PAGE. After transferring onto the membrane the blots were probed with anti-PI3K. Actin was used as a loading control. Band intensities were analyzed by densitometry scanning using image. The data point represent x?±s of relative optical density (n=3). # P<0.01 vs. control."

Figure 3

Treatment with 50 Hz 0.6 mT pulse electromagnetic fields (PEMF) enhances the expression of p-AKT The protein level was examined by Western blotting. Protein in lysates were separated by 12% SDS-PAGE. After transferring onto the membrane the blots were probed with anti-p-AKT. Band intensities were analyzed by densitometry scanning using image. The data points represent x?±s of relative optical density (n=3). # P<0.01 vs. control."

Figure 4

Effect of pulse electromagnetic fields (PEMF) and LY294002 on ALP activity in rat osteoblast cells The ALP activity of osteoblast cultures was measured using an ALP assay kit at one day (1.5 h/d) after PEMF treatment. Osteoblasts were seeded at 1×105 cells/mL in 96-well plate. They were sub-cultures and randomly divided into four group: Control group, PEMF group (treated by 50 Hz 0.6 mT PEMF), LY294002 group (treated with 50 μmol/L LY294002), and LY294002+PEMF group (treated with 50 μmol/L LY294002 and 50 Hz 0.6 mT PEMF). The data represent x?±s (n=3). # P<0.01 vs. control (Con);☆ P<0.01 vs. PEMF."

Figure 5

Effect of pulse electromagnetic fields (PEMF) and LY294002 on gene expression in rat osteoblast cells After treated by PEMF for 3 days (1.5 h/d), osteoblasts were washed with PBS and total RNA was isolated using RNAisoTM Plus reagent, mRNA expression of Bmp-2 (A), Collagen-1 (B) and Osterix (C) was quantitated by RT-PCR. The GAPDH was used as internal control. The data represent x ? ±s of mRNA expression level (n=3). *P<0.05, # P<0.01, vs. control (Con); △P<0.05, ☆P<0.01, vs. PEMF. "

Figure 6

siRNA transfection interferes IFT88 gene and protein expression The inhibition of IFT88 gene expression as revealed by RT-PCR (A), and the protein expression inhibition as revealed by Western blot (B and C) in siRNA group compared to negative control. SC, scrambled control. #P<0.01, vs. SC."

Figure 7

Effects of IFT88 siRNA transfection on primary cilia of rat osteoblasts A, normal primary cilia as immunofluorescence stained by acetylated-tubulin (green); B, primary cilia became dotted and short after 24 h of IFT88 siRNA transfection; C, percentages of cells with primary cilia in negative control and siRNA groups. SC, scrambled control. # P<0.01, vs. SC."

Figure 8

Effect of PEMF and RNAi on the protein expression of PI3K The protein level was examined by Western blot. Protein in lysates were separated by 12% SDS-PAGE. After transferring onto the membrane the blots were probed with anti-PI3K. Actin was used as a loading control. Band intensities were analyzed by densitometry scanning using image. The data point represent x ? ±s of relative optical density (n=3). SC, scrambled control. *P<0.05, vs. SC; ☆P<0.01, vs. SC+PEMF. "

Figure 9

Effect of PEMF and RNAi on ALP activity in rat osteoblast cells The ALP activity of osteoblast cultures was measured using an ALP assay kit at one day (1.5 h/d) after PEMF treatment. Osteoblasts were seeded at 1×105cells/mL in 96-well plate. They were sub-cultures and randomly divided into four group: SC group (transfected with scrambled control siRNA), SC+PEMF group (transfected with scrambled control siRNA and treated by 50 Hz 0.6 mT PEMF), siRNA group(transfected with IFT88 siRNA), and siRNA+PEMF group (transfected with IFT88 siRNA and treated by 50 Hz 0.6 mT PEMF). The data represent x ? ±s of absorbance value±standard deviation (n=3). SC, scrambled control. #P<0.01, vs. SC;☆P<0.01, vs. SC+PEMF. "

Figure 10

Effect of PEMF and RNAi on osteogenic gene expression in rat osteoblast cells After treated by PEMF for 3 days (1.5 h/d), osteoblasts were washed with PBS and total RNA was isolated using RNAisoTM Plus reagent, mRNA expression of Bmp-2 (A), Collagen-1 (B) and Osterix (C) was quantitated by RT-PCR. The GAPDH was used as internal control. The data represent x ? ±s of mRNA expression level±standard deviation (n=3). SC, scrambled control. *P<0.05, # P<0.01 vs. SC; △P<0.05, ☆ P<0.01 vs. SC+PEMF. "

Figure 11

Effects of IFT88 siRNA-mediated primary cilium inhibition on PEMF-induces promotion on mineralization of rat calvarial osteoblasts A, the Alizarin red staining results after 13 d; B, quantification of mineral matrix deposition using Image-Pro Plus 6.0 program. Results shown are the average of three independent tests. Each red dot represents a calcified nodule unit. Large numbers of red dots indicate more active osteogenic differentiation. C, the number of calcified nodules quantified by Image-Pro Plus 6.0. The data points represent x ? ±s of calcified tubercle area level (n=3). SC, scrambled control. # P<0.01, vs. SC, ☆P<0.01, vs. SC+PEMF. "