Figure 6.

Apoptosis of growth plate chondrocytes treated with various concentrations of extracellular P_i in the absence or presence of RA and PFA. (A) Cell number of growth plate chondrocytes cultured in the presence of 1, 4, and 8mM extracellular P_i for 4 days. Cell number was determined using the CCK-8 assay; cell number of cells cultured in the presence of 1mM P_i was set to 100%. Data are means of four experiments; error bars represent standard deviations (^ap < 0.01 vs. 1mM P_i-treated cells). (B) The levels of bcl-2 mRNA were determined after 2-day treatment with various P_i concentrations (1, 2.5, 4, 8mM) in the absence (− PFA) or presence of PFA (+ PFA) by real-time PCR and SYBR Green and normalized to the 18S RNA levels. Data are means of triplicate PCRs using RNA from three different cultures; error bars represent standard deviations. (C) Caspase-3 activity of growth plate chondrocytes cultured in the presence of various concentrations of extracellular P_i (1, 4, 8mM) and in the absence of RA and PFA (−RA/−PFA), presence of RA and absence of PFA (+RA/−PFA), or presence of RA and PFA (+RA/+PFA) for 4 days. Caspase-3 activity was measured and normalized to the total protein concentration. Data are means of four experiments; error bars represent standard deviations (^ap < 0.01 vs. 1mM P_i-treated cells; ^bp < 0.01 vs. 4mM P_i-treated cells; ^cp < 0.01 vs. 1mM P_i/RA-treated cells; ^dp < 0.01 vs. 4mM P_i/RA-treated cells; ^ep < 0.01 vs. 8mM P_i/RA-treated cells). (D) Percent TUNEL-positive cells among growth plate chondrocytes cultured as in (C) as determined by flow cytometric analysis. Data are means of four experiments; error bars represent standard deviations (^ap < 0.01 vs. 1mM P_i-treated cells; ^bp < 0.01 vs. 4mM P_i-treated cells; ^cp < 0.01 vs. 1mM P_i/RA-treated cells; ^dp < 0.01 vs. 4mM P_i/RA-treated cells; ^ep < 0.01 vs. 8mM P_i/RA-treated cells).