Mice
Cell lines
Analysis of bone phenotype
Plasmids
Antibodies
Cellular fractionation
Immunofluorescence staining
Coimmunoprecipitation to analyze association with p300
RNA extraction and real-time reverse transcription polymerase chain reaction analysis
Primer sequences used for PCR
Luciferase reporter assay
RNA interference
Measurement of intracellular ROS level
FSS experiments
ChIP assay
Hemilateral hindlimb unloading experiments
Statistical analysis
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.