MLO-Y4 osteocytes were exposed to FSS of 1 Pa at a frequency of 1 Hz, as described previously (47) with slight modifications. MLO-Y4 osteocytes were cultured in a 60-mm plastic dish (Sumitomo Bakelite, Akita, Japan) precoated with 0.1% bovine gelatin (Sigma-Aldrich). The experiments were conducted 1 or 2 days after the cells were seeded. To expose MLO-Y4 cells to FSS, a parallel plate flow chamber and roller pump (HV-07523-80, Masterflex) were used. The flow chamber, which was composed of a cell culture dish, a polycarbonate I/O unit, and a silicone gasket, generated a 35-mm-long, 26.7-mm-wide, 0.4-mm-high flow channel. The system was placed in a CO2 incubator to maintain pH and temperature of culture medium.

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