Himac cf7d2

ATDC5 cells were cultured in the incubator at 37°C for 4 days to form monolayers and then exposed to hypergavity for a certain time using a centrifuge (Himac CF7D2; Hitachi). The gravity loaded was calculated as shown in the following equation; Gravity = 1118 × (radius) × (RPM)² × 10⁻⁸. Control samples were also prepared and kept in the same conditions without hypergravity.
We first loaded 32 G of hypergravity to the ATDC5 monolayers 1 hour daily for 3 days. Since c-fos is known to be an early response gene to biochemical and mechanical stimuli, we first performed a time-course experiment for short periods (0, 30, 60, and 120 min) of exposure to hypergravity, followed by a dose-response experiment by modulating the intensity of hypergravity (18.7, 33.3, 52.0, 207.9, and 467.9 G). Based on the results of both the time-course and dose-response experiments, inhibitor tests were carried out for real-time PCR, staining and western blotting.

The effect of pH on caseinolytic activity was determined as follows. Casein (Hammerstein casein [Merck]) solution (0.3%) was prepared in 50 mM Britton-Robinson buffer (50 mM phosphoric acid/acetic acid/boric acid; with pH adjusted with NaOH) at pH values ranging from 6 to 10, and 200 μL of 0.3% casein solution was dispensed into the wells of a 96-well Flat-Bottom Assay Plate (Iwaki). After preincubation at 30 °C for 10 min in a Bio Shaker M/BR-024 (Taitec), a suitably diluted solution of enzyme sample (20 μL) was added and mixed gently by shaking in the Bio Shaker M/BR-024 at 30 °C for 15 min. The reaction was stopped by addition of 100 μL of 5% trichloroacetic acid. Next, 300 μL of the mixture was transferred into a Filter plate MultiScreen-HV (Merck Millipore) and centrifuged at 2500 rpm using a himac CF7D2 (Hitachi) to remove denatured protein. Subsequently, 200 μL of the filtrate was transferred into the wells of a UV Flat-Bottom Microtiter Plate (Thermo Fisher Scientific). The peptide concentration of the filtrate was determined by monitoring at 280 nm using an Infinite M200 microplate reader (Tecan). One unit (U) of caseinolytic activity was defined as the amount of enzyme needed to produce acid-soluble peptide equivalent to 1 μmol of L-Tyr per min.