FIGURE 7.

Expression of Has2 is suppressed by nSMase2 via the PI3K or Akt pathway in ATDC5 cells, whereas localization of nSMase2 and Has2 is mutually exclusive in the growth plate cartilage of mouse embryo. A and B, ATDC5 cells (A) or mouse primary chondrocytes (B) were transfected with control siRNA (siCont) or Has2 siRNA (siHas2) for 16 h and then treated with BMP-2 (300 ng/ml) for 6 days. Quantitative RT-PCR analysis was performed for Col2a1 and Col10a1. C, ATDC5 chondrocytes were transfected with control siRNA (siCont) or Smpd3 siRNA (siSmpd3) for 16 h and then treated with BMP-2 (300 ng/ml) for 6 days. Quantitative RT-PCR analysis was performed for Has1, Has2, and Has3. D, immunofluorescence for nSMase2 or Has2 was performed in ATDC5 chondrocytes. IgG was used as negative control. Scale bar, 50 μm. E, immunofluorescence for nSMase2 or Has2 was performed on mouse primary chondrocytes. Biotin-conjugated hyaluronan-binding protein (HABP) and Alexa Fluor 488-conjugated streptavidin were applied to detect hyaluronan. IgG was the negative control. Scale bar, 50 μm. F, expression of nSMase2 or Has2 in mouse E17.5 humerus cartilage was evaluated by immunofluorescence. Biotin-conjugated HA-binding protein and Alexa Fluor 488-conjugated streptavidin were used to detect hyaluronan. IgG was the negative control. r, resting chondrocytes; p, proliferating chondrocytes; ph, prehypertrophic chondrocytes; h, hypertrophic chondrocytes. Scale bar, 250 μm. G, ATDC5 cells were transfected with control siRNA (siCont) or Smpd3 siRNA (siSmpd3) for 16 h and further stimulated by BMP-2 (300 ng/ml) with or without LY294002 (LY, 1 μm) or MK2206 (MK, 1 μm) for 6 days. Expression of Has2 was evaluated by quantitative RT-PCR analysis. *, p < 0.05; **, p < 0.01; n.s., not significant.