Fig. 9

Effect of LEF1 regulated by let-7i-3p on the osteogenic differentiation of hASCs under cyclic strain (*p < 0.05, significant differences existed between these two groups). a hASCs were cotransfected with the let-7i-3p mimic and EX-LEF1 under cyclic strain for 6 days, and the levels of the following mRNAs were significantly increased compared to those in hASCs cotransfected with let-7i-3p mimic and EX-Ctrl: RUNX2, ALP, SPARC, LEF1, and β-catenin (2.10 ± 0.69-fold, p = 0.014; 2.80 ± 1.62-fold, p = 0.005; 2.06 ± 0.30-fold, p = 0.006; 3.06 ± 0.52-fold, p = 0.029; and 2.75 ± 0.68-fold, p = 0.010, repectively). b Protein blots are listed in the left column. hASCs were cotransfected with the let-7i-3p mimic and EX-LEF1 under cyclic strain for 6 days. The protein expression levels of the above markers were also increased: RUNX (22.02 ± 0.38-fold, p = 0.004), ALP (2.03 ± 0.07-fold, p = 0.002), SPARC (2.54 ± 0.60-fold, p = 0.004), LEF1 (2.91 ± 0.61-fold, p = 0.004), and β-catenin (1.49 ± 0.17-fold, p = 0.022). c The immunofluorescence intensity of β-catenin in the nucleus was enhanced in hASCs cotransfected with the let-7i-3p mimic and EX-LEF1(scale bar 100 μm). d The protein expression levels of cytoplasmic and nuclear β-catenin in hASCs cotransfected with the let-7i-3p mimic and EX-LEF1 were respectively increased 1.30 ± 0.10-fold (p = 0.026) and 1.40 ± 0.15-fold (p = 0.017) than those in the group transfected with the let-7i-3p mimic and EX-Ctrl. e hASCs were cotransfected with the let-7i-3p inhibitor and siLEF1 under cyclic strain for 6 days. The mRNA levels of the above markers were decreased by 1.52 ± 0.26-fold (p = 0.026), 3.12 ± 1.55-fold (p = 0.002), 2.55 ± 0.25-fold (p = 0.038), 3.92 ± 0.72-fold (p = 0.018), and 2.25 ± 1.24-fold (p = 0.030), respectively, compared to those in hASCs cotransfected with the let-7i-3p inhibitor and siR-Ctrl. f Protein blots are listed in the left column. hASCs were cotransfected with the let-7i-3p inhibitor and siLEF1 under cyclic strain for 6 days. The protein expression levels of the above markers were also decreased 2.00 ± 0.42-fold (p = 0.002), 1.54 ± 0.17-fold (p = 0.013), 1.71 ± 0.19-fold (p = 0.003), 2.24 ± 0.28-fold (p = 0.001), and 1.55 ± 0.06-fold (p = 0.012), respectively. g The immunofluorescence intensity of β-catenin in the hASC nucleus was weakened in hASCs cotransfected with the let-7i-3p inhibitor and siβ-catenin (scale bar 100 μm). h The protein expression levels of cytoplasmic and nuclear β-catenin in hASCs cotransfected with the let-7i-3p inhibitor and siLEF1were respectively decreased 1.41 ± 0.19-fold (p = 0.036) and 2.13 ± 0.35-fold (p = 0.001) than those in the group transfected with the let-7i-3p inhibitor and siR-Ctrl. i hASCs were cotransfected with the let-7i-3p mimic and EX-β-catenin under cyclic strain for 6 days: compared to those in the group transfected with the let-7i-3p mimic and EX-Ctrl, the mRNA expression levels of RUNX2, ALP, SPARC, and β-catenin were increased 2.42 ± 0.92-fold (p = 0.004), 4.27 ± 1.78-fold (p = 0.002), 3.06 ± 0.41-fold (p = 0.001), and 3.17 ± 1.09-fold (p = 0.008); the mRNA expression levels of LEF1 was decreased 1.47 ± 0.21-fold (p = 0.016), respectively. j Protein blots are listed in the left column. hASCs were cotransfected with the let-7i-3p mimic and EX-β-catenin under cyclic strain for 6 days: compared to those in the group transfected with the let-7i-3p mimic and EX-Ctrl, the protein expression levels of RUNX2, ALP, SPARC and β-catenin were increased 1.57 ± 0.06-fold (p = 0.011), 2.10 ± 0.03-fold (p = 0.001), 3.16 ± 1.01-fold (p = 0.002), and 1.50 ± 0.04-fold (p = 0.006); the protein expression levels of LEF1 were decreased 1.40 ± 0.13-fold (p = 0.023), respectively. k The immunofluorescence intensity of β-catenin in the hASC nucleus was enhanced in hASCs cotransfected with the let-7i-3p mimics and EX-β-catenin (scale bar 100 μm). l The protein expression levels of cytoplasmic and nuclear β-catenin in hASCs cotransfected with the let-7i-3p mimic and EX-β-catenin were respectively increased 1.35 ± 0.12-fold (p = 0.019) and 2.23 ± 0.27-fold (p = 0.008) than those in the group transfected with the let-7i-3p mimic and EX-Ctrl. m hASCs were cotransfected with the let-7i-3p mimic and EX-β-catenin under cyclic strain for 6 days: compared to those in the group transfected with the let-7i-3p inhibitor and siβ-catenin, compared to those in the group transfected with the let-7i-3p inhibitor and siR-Ctrl, the mRNA levels of RUNX2, ALP, SPARC, and β-catenin were decreased 1.73 ± 0.24-fold (p = 0.015), 2.63 ± 0.69-fold (p = 0.004), 2.36 ± 0.46-fold (p = 0.006), and 3.75 ± 0.21-fold (p = 0.001); the mRNA expression levels of LEF1 was increased 1.89 ± 0.74-fold (p = 0.009), respectively. n Protein blots are listed in the left column. hASCs were cotransfected with the let-7i-3p mimic and EX-β-catenin under cyclic strain for 6 days: compared to those in the group transfected with the let-7i-3p inhibitor and siR-Ctrl, the protein expression levels of RUNX2, ALP, SPARC, and β-catenin were decreased 1.48 ± 0.15-fold (p = 0.007), 2.12 ± 0.14-fold (p = 0.006), 1.40 ± 0.01-fold (p = 0.020), and 1.51 ± 0.17-fold (p = 0.011); the protein expression levels of LEF1 were increased 1.53 ± 0.19-fold (p = 0.006), respectively. o The immunofluorescence intensity of β-catenin in the hASC nucleus was weaken in hASCs cotransfected with the let-7i-3p inhibitor and siβ-catenin (scale bar 100 μm). p The protein expression levels of cytoplasmic and nuclear β-catenin in hASCs cotransfected with the let-7i-3p inhibitor and siβ-catenin were respectively decreased 1.35 ± 0.09-fold (p = 0.006) and 1.83 ± 0.04-fold (p = 0.004) than those in the group transfected with the let-7i-3p inhibitor and siR-Ctrl