FIGURE 4.

LIPUS-induced ERK phosphorylation is mediated by Cot/Tpl2 activation. A, ST2 cells were cultured in adipogenic differentiation medium (dexamethasone, insulin, and IBMX). Cells were treated with or without 2.5 mm U0126 for 60 min and stimulated by LIPUS for 20 min every day. After each LIPUS treatment, cell culture media were changed to remove U0126. 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). B, ST2 cells were cultured in osteogenic differentiation media (l-ascorbic acid 2-phosphate trisodium and β-glycerophosphate) with or without 2.5 mm U0126 for 60 min and stimulated by LIPUS for 20 min every day. After each LIPUS treatment, cell culture media were changed to remove U0126. The expressions of osteogenic marker genes were analyzed as in A. C, 3T3-L1, MC3T3-E1, and ST2 cells were stimulated by LIPUS for 20 min. Cells were lysed in RIPA lysis buffer immediately after the stimulation. Cell lysates were separated by SDS-PAGE, and levels of phosphorylated and total Cot/Tpl2 proteins were determined by Western blotting. D, 3T3-L1, MC3T3-E1, and ST2 cells were pretreated with 5 μm TKI (a Cot/Tpl2-specific inhibitor) for 30 min followed by LIPUS stimulation for 20 min. Cells lysates were prepared in RIPA lysis buffer and separated by SDS-PAGE. The levels of phosphorylated and total ERK proteins were determined by Western blotting. E and F, 3T3-L1, MC3T3-E1, and ST2 cells were transiently transfected with either Cot/Tpl2 siRNA or control siRNA. The effects of Cot/Tpl2 siRNA on Cot/Tpl2 protein expression levels (E) and LIPUS-induced ERK phosphorylation (F) were confirmed by Western blotting. mW, milliwatts.