Osteoclastogenesis Molecular Mechanism Analysis

RAW264.7 cells were seeded at 1.0 × 10⁵ /well in six-well plates with 50 ng/mL RANKL for 3 or 5 days. Total RNA was extracted using the ReliaPrep™ RNA Cell Miniprep System (Promega, Madison, WI, USA). Total RNA (0.5 μg) was reverse-transcribed using the SuperScript® VILO™ cDNA Synthesis Kit (Invitrogen ThermoFisher Scientific) according to the manufacturer’s instructions. Real-time PCR was performed using SYBR Premix Ex Taq II (TaKaRa Bio, Shiga, Japan) and gene-specific primers with the Thermal Cycle Dice Real-Time System TP800 (TaKaRa Bio). Reactions were performed in duplicate, and relative mRNA levels were calculated by the comparative threshold cycle method using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the internal control. The primer sequences were as follows: GAPDH (forward, 5′-TGTGTGTCCGTCGTGGATCTGA-3′; reverse, 5′-TTGCTGTTGAAGTCGCAGGAG-3′^{35 (link)}); TRPV2 (forward, 5′-AGGAGCTGACTGGACTGCTA-3′; reverse, 5′-GAGCCTTCTGTGTATGCCGA-3′^{28 (link)}); TRPV4 (forward, 5′- CCACCCCAGTGACAACAAG-3′; reverse, 5′- GGAGCTTTGGGGCTCTGT-3′^{28 (link)}); NFATc1 (forward, 5′-CCCGTCACATTCTGGTCCAT-3′; reverse, 5′-CAAGTAACCGTGTAGCTGCACAA-3′^{36 (link)}); Cathepsin K (forward, 5′-AGGCAGCTAAATGCAGAGGGTACA-3′; reverse, 5′-AGCTTGCATCGATGGGACACAGAGA-3′^{37 (link)}); and TRAP (forward, 5′-AGCTTGCATCGATGGGACACAGAGA-3′; reverse, 5′-GTCAGGAGTGGGAGCCATATG-3′^{37 (link)}).