Sybr green

MA (purity >98%) was obtained from the National Institutes for Food and Drug Control (Beijing, China) and dissolved in dimethyl sulfoxide (DMSO) at a stock concentration of 100 mmol L⁻¹. Alpha modified minimal essential medium (α‐MEM), penicillin/streptomycin (P/S), and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific (Carlsbad, CA, USA). M‐CSF and GST‐rRANKL were produced in our laboratory according to previous reports.25, 26 Tetrazolium salt (MTS) solution, TRIzol, and rhodamine‐conjugated phalloidin were purchased from Thermo Fisher Scientific (San Jose, CA, USA). Oligo‐dT primer and SYBR Green were obtained from Imgenex (Littleton, CO, USA). Primary antibodies specific for NFATc1, integrin β3, Cathepsin K, V‐ATPase‐d2, IκB‐α, pERK1/2, ERK1/2, and β‐actin were obtained from Santa Cruz Biotechnology (San Jose, CA, USA). Antibodies specific for c‐Fos, pJNK1/2, JNK1/2, p‐p38, and p38 were purchased from Cell Signaling Technology (Beverly, MA, USA).

A total of 5 × 10³ BMMs per well were plated in a 6‐well plate in the presence of GST‐rRANKL (50 ng/mL) and M‐CSF (50 ng/mL) with or without various densities of MA for 5 days. As described in our previous study,32 TRIzol (Qiagen, Hilden, Germany) was used to extract total RNA from cells. With an oligo‐dT primer, single‐stranded cDNA was reverse transcribed from 2 μg total RNA. The resulting cDNA was then used for real‐time PCR based on SYBR Green (Imgenex, Littleton, CO, USA) with the specific primers displayed in Table 1. The expression level was normalized to Hmbs expression. The fold change was determined using the Livak equation, and the ratios compared to the vehicle group were calculated.

BMMs at 2 × 10⁴ cells per well were plated in a six-well plate and treated with GST-rRANKL (50 ng/ml) and M-CSF (25 ng/ml) in the presence or absence of PA at various concentrations for 5 days. Total RNA was extracted using TRIzol. An oligo-dT primer was used to reverse transcribe single-stranded cDNA from 2 μg of total RNA. Real-time PCR amplification of the resulting cDNA was performed using SYBR Green (Imgenex, Littleton, CO, United States) and specific primers. The expression levels of target genes were normalized to expression of the housekeeping gene Hprt. The Livak equation was used to calculate the fold change in expression and ratio of expression in the experiment group compared to the vehicle group. The sequences of all the primers used are listed in Table 1.