Fig. 7

TGF-β signaling was involved in the regulation of LHX9 and metastasis-related proteins. a U2OS or Saos-2 cells were cultured without or with 1, 5, 10 ng/ml TGF-β1 for 24 h (left two panels), or cultured without or with 5 ng/ml TGF-β1 for 24, 48 and 72 h, respectively (right two panels). The expression of FRS2 protein was measured by Western blot, β-actin was used as the loading control. The normalized protein expression levels against β-actin were shown in the below histograms. b U2OS or Saos-2 cells were cultured without or with 1, 5, 10 ng/ml TGF-β1 for 24 h, then the relative expression of LHX9 was assessed by RT-qPCR. The mRNAs were normalized to GAPDH, experiments were performed in triple. c U2OS or Saos-2 cells were cultured without or with 5 ng/ml TGF-β1 for 24, 48 and 72 h, respectively, and the relative expression of LHX9 was measured by Western blot. GAPDH was used as the loading control. The normalized expression of LHX9 against GAPDH was shown in the right histograms. d U2OS or Saos-2 cells were transfected with shNC or shFRS2, then they were cultured for 48 h. The relative expression of TGF-beta and TGF-beta R1 was assessed by RT-qPCR. The mRNAs were normalized to GAPDH mRNA (n = 3). e U2OS or Saos-2 cells were transfected with shNC or shFRS2, then they were cultured in the absence or presence of TGFβR1 inhibitor (SB431542) for 48 h. The expression of LHX9, COL1A1, MMP-9, and MMP-1 was measured by Western blot. GAPDH was used as the loading control. The normalized expression of these proteins against GAPDH was shown in the right histograms. a–e The result was a representative of three independent experiments. Error bars represented mean ± SD. p values were determined by one-way analysis of variance (ANOVA) followed by Tukey post hoc test (a–c, e) or unpaired two-tail Students’ t-test (d). **p < 0.01, *p < 0.05. NS not significant