FIGURE 3.

Role of pro-apoptotic Bcl-2 family member proteins in P_i-induced cell death. A, expression patterns of pro-apoptotic Bcl-2 family member proteins in ATDC5 cells during ITS treatment as determined by time and RT-PCR. The results are expressed as the means ± S.D. of three cultures. Experiments were repeated at least three times, and the representative data are presented. ^*, significantly different from day 0, p < 0.05. B, expression patterns of pro-apoptotic Bcl-2 family member proteins in ATDC5 cells after P_i stimulation as determined by real time PCR. ATDC5 cells were pretreated with ITS for 14 days (0 h) before P_i stimulation (20 mm). The results are expressed as the means ± S.D. of three cultures. Experiments were repeated at least three times, and the representative data are presented. ^*, significantly different from time 0, p < 0.05. C, effect of gene silencing of pro-apoptotic Bcl-2 family member proteins on cell viability of primary chondrocytes 24 h after P_i stimulation. Gene silencing of Bnip3 and Nix significantly promoted the cell viability, p < 0.05. The results are expressed as the means ± S.D. of six cultures. Experiments were repeated at least three times, and the representative data are presented. GFP, green fluorescent protein. D, Western blot analysis after P_i stimulation. Protein levels of Bnip3 increased in ATDC5 cells after 6 h of P_i stimulation, which was followed by caspase-7 activation. Bnip3 was detected as two bands. E, immunohistological examination of a murine metatarsal bone (E18.5) with anti-Bnip3 antibody. Bnip3 expression was primarily localized in the hypertrophic layers of chondrocytes.