FIGURE 6.

Cot/Tpl2 is an essential signaling molecule of LIPUS-induced suppression of adipogenesis and promotion of osteogenesis. A, ST2 cells were cultured in adipogenic differentiation media (dexamethasone, insulin, and IBMX) with or without 5 μm TKI and stimulated by daily LIPUS for 20 min. After 15 days, cells were stained with oil red O to determine lipid droplet appearance. B, ST2 cells were induced to differentiate as in A. After 15 days, total RNAs were isolated, reverse transcribed, and analyzed by real time PCR. Each experiment was repeated at least three times with consistent results. Relative mRNA expression levels in comparison with Rpl13a mRNA are shown. Error bars represent S.D. Statistical significance was determined by Student's t test (*, p <0.05; **, p < 0.01). C, ST2 cells were cultured in osteogenic differentiation media (280 μm l-ascorbic acid 2-phosphate trisodium and 5 mm β-glycerophosphate) with or without 5 μm TKI and stimulated by daily LIPUS for 20 min. After 21 days, cells were stained with alizarin red S for the detection of calcification. The calcified area was photographically measured, and the mineralization ratio relative to control was expressed as mean ± S.D. Statistical significance was determined by Student's t test (**, p < 0.01). D, ST2 cells were induced to differentiate as in C. Total RNAs were isolated and reverse transcribed. The gene expressions of osteogenic markers were analyzed by real time PCR. Each experiment was repeated at least three times with consistent results. Relative mRNA expression levels in comparison with Rpl13a mRNA are shown. Error bars represent S.D. Statistical significance was determined by Student's t test (**p < 0.01).