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Fig. 3 | Cell Communication and Signaling

Fig. 3

From: Pasteurella multocida toxin- induced osteoclastogenesis requires mTOR activation

Fig. 3

Osteoclast differentiation of RAW264.7 cells with PMT requires mTOR activation and shows a distinct morphology. (a) RAW264.7 cells were stimulated with PMT (5 nM) or RANKL (50 ng/ml) as a positive control for osteoclast differentiation. After 4 days of stimulation, cells were labelled in an enzymatic reaction for the activity of the enzyme TRAP (ELF97) in green. Nuclei were stained with DAPI (blue) and the cytoskeleton with TRITC-Phalloidin (red). Pictures were taken by confocal laser scanning microscopy and representative pictures are shown (number of independent experiments n ≥3). (b) RAW264.7 cells were seeded in 6-well plates, stimulated with 5 nM PMT or 50 ng/ml RANKL for 24 h-48 h before preparation of cDNAs. Quantitative RT-PCR was performed and the graphs display the relative expression for Oscar, Nfatc1, Ctsk (cathepsin K), Acp5 (TRAP) and ATP6v0d2 normalized to S29 expression. The indicated standard deviation was obtained from four experiments (mean ± SD; n = 4 and n = 3 for Nfatc1). Statistical analysis was performed using an unpaired Student’s t-test comparing gene expression to the untreated sample (*: p ≤ 0.05; **: p ≤ 0.005; ***:p ≤ 0.0005). (c) The numbers of conventionally TRAP-stained cells were quantified after 3 days of stimulation with the indicated concentrations of PMT or 50 ng/ml RANKL in the presence or absence of rapamycin (10 ng/ml). The graph displays the number of TRAP-positive, multinucleated (≥3) cells. The indicated standard deviation was calculated from three independent experiments (mean ± SD; n = 3). Statistical analysis was performed using an unpaired Student’s t-test (***: p ≤ 0.0005)

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