Oxidative Medicine and Cellular Longevity / 2013 / Article / Fig 3

Research Article

Tanshinol Attenuates the Deleterious Effects of Oxidative Stress on Osteoblastic Differentiation via Wnt/FoxO3a Signaling

Figure 3

Tanshinol counteracts inhibitory effect of oxidative stress on osteoblastic differentiation. (a) C2C12 cells were treated with increasing concentrations of BMP-2 for 24 h to determine the optimal concentration using qRT-PCR. Cells were cotreated with BMP-2 (100 ng/mL) and Tanshinol (1 μM) or Resveratrol (1 μM) in the presence or absence of H2O2 (200 μM), and determinations were made as follows. (b) Cells were stained using ALP staining Kit, and (c) the percentage of positively stained cell populations was counted by Image J software to analyse the extent of osteoblastic differentiation at day 3; (d) ALP activity in the cell lysates and (e) osteocalcin secretion in the supernatant of media were measured by ALP Assay Kit and ELISA Kit at day 3, respectively; (f) mineralization activity with the indicated treatments was stained using alizarin red S at day 10. Note: (1) Con (vehicle control); (2) B (BMP-2); (3) B + T (BMP-2 + Tanshinol); (4) B + H (BMP-2 + H2O2); (5) B + H + T (BMP-2 + H2O2 + Tanshinol); (6) B + H + R (BMP-2 + H2O2 + Resveratrol). Data shown are the mean ± SEM of at least three independent experiments. versus vehicle control and versus BMP-2 + H2O2 treatment. Original magnification ×100 in (b).

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