Fig. 4

PMT activates targets known to be important for osteoclast differentiation. RAW264.7 cells were left untreated or stimulated with PMT (5 nM) w or w/o rapamycin (10 ng/ml). After incubation for the indicated period of times, cells were examined for Akt (Thr308) phosphorylation or the total amount of Akt (a) and PDCD4 (b). Shown are representative data from three independent experiments. (c) Pdcd4 expression (mean ± SD; n = 2) was determined by RT-PCR in the presence or absence of rapamycin (10 ng/ml) using cells treated with 5 nM PMT or 50 ng/ml RANKL for 6 h (n = 2). (d) PDCD4 siRNA transfection was performed according to the manufacturer’s protocol. RT-PCR of Pdcd4 expression was determined after 36 h and was normalized to S29 expression (n = 3). Statistical analysis was performed using a paired Student’s t-test (*: p ≤ 0.05). (e) 24 h after siRNA transfection, cells were stimulated for 12 h with PMT (5 nM) in the presence or absence of 10 ng/ml rapamycin. The expression of Ctsk was monitored by RT-PCR (mean ± SD; n = 2). Analysis for (d) and (e) was performed using the same samples of cDNA (*: p ≤ 0.05). (f ) Phosphorylation of c-Jun (Ser63) and nuclear localisation of c-Jun (g) was detected by western blot analysis. Shown are representative data from three independent experiments