MC3T3-E1 cells were transfected with Cntnap4 shRNA Lentiviral particles (Santa Cruz Biotechnology, Dallas, TX, USA) or non-target control shRNA by Lipofectamine 3000. The positive transfected colonies were selected by Puromycin (MilliporeSigma).
Control and stable Cntnap4-knockdown (Cntnap4-KD) MC3T3-E1 cells were seeded on 24-well plates for alkaline phosphatase (ALP), Alizarin red, and IHC staining. In addition, cells were seeded on 6-well plates for osteogenic genes expression assay. Both cell types were cultured in osteogenic differentiation medium (α-MEM, 10% fetal bovine serum [FBS; Thermo Fisher Scientific], 50 μg/mL ascorbic acid, and 10 mM β-glycerophosphate [MilliporeSigma]) with or without 500 ng/mL Nell-1 or 100 ng/mL BMP2 (Medtronic, Minneapolis, MN, USA). Protein isolation and Western blot were performed as previously described.(28 (link))
Control and stable Cntnap4-knockdown (Cntnap4-KD) MC3T3-E1 cells were seeded on 24-well plates for alkaline phosphatase (ALP), Alizarin red, and IHC staining. In addition, cells were seeded on 6-well plates for osteogenic genes expression assay. Both cell types were cultured in osteogenic differentiation medium (α-MEM, 10% fetal bovine serum [FBS; Thermo Fisher Scientific], 50 μg/mL ascorbic acid, and 10 mM β-glycerophosphate [MilliporeSigma]) with or without 500 ng/mL Nell-1 or 100 ng/mL BMP2 (Medtronic, Minneapolis, MN, USA). Protein isolation and Western blot were performed as previously described.(28 (link))
