Inhibition of Osteoclast Activation by Phloretin through Disturbing αvβ3 Integrin-c-Src Pathway

Figure 7

Inhibition of RANKL-induced Syk activation (a) and PLCγ-Akt signaling (b) by phloretin, and actin ring formation by PI3K inhibition (c). Raw 264.7 macrophages were cultured in α-MEM and exposed to 50 ng/mL RANKL for 5 days in the absence and presence of 1–20 μM phloretin. Cell lysates were subject to SDS-PAGE and Western blot analysis with a primary antibody against Syk, phospho-Syk, PLCγ, Akt, and phospho-Akt. Representative blot data were obtained from three independent experiments, and β-actin protein was used as an internal control. The bar graphs (mean ± SEM) in the bottom panels represent quantitative results of blots obtained from a densitometer. Respective values not sharing a common letter are different at . Raw 264.7 cells were cultured for 5 days with 50 ng/mL RANKL in the presence of 20 μM phloretin and 10 μM LY294002 (PI3K inhibitor). Differentiated cells were fixed in 4% paraformaldehyde for 10 min and fluorescent rhodamine phalloidin was added to fixed cells. Fluorescent images were taken with a fluorescence microscope. Original magnification of microscopic images (), 400-fold.

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