Muscle, connective tissue, and periosteum were removed from femurs and tibiae to separate osteocytes from whole bone, and the bones were cut at the metaphyses. Hematopoietic cells and osteoblastic cells were removed from the diaphyses of femurs and tibiae by flushing with PBS and using a microinterdental brush (0.2 mm in diameter). The remaining bone was used as a source of osteocyte-enriched cells. The osteocyte-rich bones were frozen in liquid nitrogen and ground into a powder using a tissue pulverizer. The powdered bone was then subjected to extraction of RNA, DNA, and protein. MLO-Y4 osteocytes, MEFs, HEK293 cells, and osteocyte fractions were harvested, and cell lysates were biochemically analyzed.

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