Fig 2: Smad1/5 signaling regulates 20 dynes/cm2 shear force-induced SCD-1 expression in human MG63 osteosarcoma cells. (A) Cells were kept as the controls (CL) or stimulated with 20 dynes/cm2 shear force for 1, 4, 8 and 24 h, and their Smad1/5 phosphorylation was analyzed by Western blot. (B,C) Cells were transfected with control-, Smad1-, or Smad5-specific siRNA (siCL, siSmad1, siSmad5) and then kept as the controls (CL) or stimulated with 20 dynes/cm2 shear force (SS) for 8 h. (B) The SCD-1 protein expression was analyzed by Western blot. (C) The Smad1 or Smad5 gene expressions were analyzed by real-time quantitative PCR. Results in (A,B) are representative of three independent experiments with similar results. Statistical data in (A–C) are mean ± SEM from three independent experiments. Protein phosphorylation and expression (A,B) and mRNA expression (C) levels were normalized to Smad1/5/β-actin and GAPDH, respectively. * p < 0.05 versus untreated control cells (CL), siCL-CL, or siCL. # p < 0.05 versus siCL-SS-treated cells.

Fig 3: Smad5-p75NTR signaling regulates BMP7-promoted human LN18 glioblastoma cell transmigration and migration. Human LN18 glioblastoma cells were transfected with control-, Smad5- or p75NTR-specific siRNA and then kept as the controls or treated with BMP7 (50 ng/mL) for 24 h. (A–C) The transmigration capabilities (A), p75NTR expression (B), and migration capabilities (C) of human LN18 glioblastoma cells were determined by the transwell assay, Western blot, and wound-healing assay, respectively. (A) *, p < 0.05 vs. siCL-CL group. #, p < 0.05 vs. siCL-BMP7 group. (B) *, p < 0.05 vs. siCL group. (C) *, p < 0.05 vs. 0 h group. #, p < 0.05 vs. siCL-24 h group. Scale bars in (A,C) are 100 μm.

Fig 4: Smad1/5 increase peroxisome proliferator-activated receptor δ (PPARδ) expression and transcription activation to regulate SCD-1 upregulation of 20 dynes/cm2 shear force stimulation in human MG63 osteosarcoma cells. (A,E) Cells were kept as the controls (CL) or stimulated with 20 dynes/cm2 shear force for 1, 4, 8 and 24 h, and their PPARs expressions (A) and PPARδ transcription activity (E) were analyzed by Western blot and transcription factor ELISA assay, respectively. (B–D,F) Cells were transfected with control- PPARα-, PPARδ-, or PPARγ-specific siRNA (siCL, siPPARα, siPPARδ, siPPARγ) (B,C) or Smad1- or Smad5-specific siRNA (siSmad1, siSmad5) (D,F) and then kept as the controls (CL) or stimulated with 20 dynes/cm2 shear force (SS) for 8 h. (B) The SCD-1 expression was analyzed by Western blot. (C) The Smad1 or Smad5 gene expressions were analyzed by real-time quantitative PCR. (D,F) The PPARδ expression (D) and transcription activity (F) were analyzed by Western blot and transcription factor ELISA assay, respectively. Results in (A,B,D) are representative of three independent experiments with similar results. Statistical data in (A–F) are mean ± SEM from three independent experiments. Protein (A,B,D) and mRNA (C) expression levels were normalized to β-actin and GAPDH, respectively. * p < 0.05 versus untreated control cells (CL), siCL-CL, or siCL. # p < 0.05 versus siCL-SS-treated cells.

Fig 5: Smad5 regulates p75NTR expression in BMP7-stimulated human LN18 glioblastoma cells. (A) Human LN18 glioblastoma cells were kept as the controls or treated with BMP7 (50 ng/mL) for 1, 4, 8, and 24 h and then the Smad1/5 phosphorylation was determined by Western blot. (B) Human LN18 glioblastoma cells were transfected with control-, Smad1- or Smad5-specific siRNA, kept as the controls or treated with BMP7 (50 ng/mL) for 24 h and then the p75NTR protein expressions were determined by Western blot. Results in (A,B) are representative of three independent experiments with similar results. Statistic data in (A,B) are mean ± SEM from three independent experiments. (A) *, p < 0.05 vs. CL group. (B) *, p < 0.05 vs. siCL-CL group; #, p < 0.05 vs. siSmad5-CL group.