Fig. 5.

Cntnap4 is indispensable for Nell-1 osteogenic bioactivity in vitro. (A) ALP staining on day 9 and Alizarin red staining on day 14 revealed increased staining in the Nell-1 and BMP2 groups of control shRNA-transfected MC3T3-E1 cells. In Cntnap4-KD MC3T3-E1 cells, high staining intensities of ALP and Alizarin red were only present in the BMP2 group. (B) A time-dependent, steady increase in Ocn and Opn staining was observed in both PBS and recombinant human Nell-1-treated control MC3T3-E1 cells. At each time point, the Nell-1-treated group demonstrated increased staining intensity when compared with the PBS-treated group. In Cntnap4-KD MC3T3-E1 cells, neither PBS nor Nell-1 treatment resulted in detectable positive staining of Ocn or Opn. (C) In Control MC3T3-E1 cells, Alp, Collagen Iα1, and Collagen Iα2 reached peak expression levels 9 days after stimulation, whereas Ocn, Opn, and Bsp displayed time-dependent patterns of increased expression. Notably, higher expression levels were detected for each gene in the Nell-1 treatment group when compared with the PBS group. However, Cntnap4-KD MC3T3-E1 cells did not exhibit significant changes in any osteogenic markers nor any differences between the PBS group and the Nell-1 group. Mean ± SEM of six independent experiments performed in triplicate is shown. *p < 0.05 when compared with the Control + PBS group. Scale bar = 100 μm.