Figure 4.

The effect of NPW‐23 on ATDC5 cell differentiation. ATDC5 cells were cultured with DMEM/F12 medium in a 12‐well plate with adensity of 50 000 cells/well. After 12 h, the cells were treated with or without NPW‐23 (200 ng/mL) containing 5% FBS and 1% ITS in DMEM/F12 medium for 4, 7, 14 and 21 d. (A,B) Alcian blue and Alizarin red staining were used to detect the proteoglycan synthesis and mineralization, respectively. (light microscopy, 10× magnification). Effects of NPW‐23 on mRNA levels of (C) p21, (D) Coll II, (E) Aggrecan, (F) Sox‐9, (G) Coll X, and (H) Runx2 in ATDC5 cells were analyzed by real‐time PCR, normalized against RPL‐19 and compared to the control group. I, Alkaline phosphatase (ALP) activity was assessed by ALP assay and described in the methods section of this paper. The data represent triplicate samples in two independent experiments. *P < 0.05 was considered significant