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. 2019 May 20;20(10):2481. doi: 10.3390/ijms20102481

Figure 4.

Figure 4

The involvement of suppression of PI3K/Akt pathway in the phloretin-induced downregulation of osteoblast differentiation markers and upregulation of adipocyte differentiation markers during BMP-2-induced osteoblastogenesis in ST2 cells. (A–C) After reaching confluence, ST2 cells were incubated in osteoblast differentiation medium for 2 days. Thereafter, the cells were treated with 100 µM phloretin for up to 12 h, total protein was extracted, and western blot analysis was performed to examine the time-dependent effect of phloretin on Akt (A). To test dose-dependency, the cells were treated with phloretin (0 to 100 µM) for 12 h (B). Quantification of the bands was performed (C). The results are representative of three experiments. Quantification results are expressed as mean ± SE (n = 3). * p < 0.05, ** p < 0.01. (D) After reaching confluence, ST2 cells were incubated in osteoblast differentiation medium with 0 or 5 µM LY294002, and von Kossa staining and Alizarin red staining were performed on day 14. (E–N) After reaching confluence, ST2 cells were incubated in osteoblast differentiation medium with 0 to 10 µM LY294002. Total mRNA was extracted on day 3, and the mRNA expression of osteoblastogenic markers (Alp, Col-1, Ocn, Runx2, and Osx) and adipogenic markers (Pparg, C/ebpa, Fas, Fabp4, and Apn) was examined by real-time PCR. The results are expressed as mean ± SE (n ≥ 5). * p < 0.05, ** p < 0.01, *** p < 0.001. Phl; phloretin, LY; LY294002.