Leukocyte acid phosphatase staining kit

After 10 days of incubation as described in Section 2.13, 4B12 cells were stained for TRAP using a leukocyte acid phosphatase staining kit (Sigma) according to the manufacturer's instructions. Cells were washed with HBSS and fixed with the provided fixing solution (18 mmol/L citrate solution; acetone and 37% formaldehyde) for 30 seconds and subsequently rinsed thoroughly with deionized water. Later, TRAP staining was performed for 1 hour at 37°C in the dark by immersion of fixed samples in 7 mg/mL Fast Garnet GBC Base solution, 0.1 M sodium nitrite solution, 2.5 mol/L acetate solution, 0.335 mol/L tartrate solution and 12.52 mg/mL naphthol AS‐BI Phosphate solution. After washing‐off the remaining fixing mixture with deionized water, cell nuclei were stained for 2 minutes using haematoxylin solution followed by rinsing in tap water. Stained samples were imaged using an inverted microscope (AxioObserverA1; Zeiss), and pictures were acquired using a Cannon PowerShot digital camera.

Bone marrow-derived mononuclear osteoclast precursors were isolated from tibiae and femurs of 6-week-old male C57BL/6J mice (Central Lab Animal, Seoul, Korea) by flushing the bone marrow cavity. Cells were treated with red blood cell lysis buffer (Sigma-Aldrich, St. Louis, MO, USA), and then incubated with minimum essential medium-alpha (α-MEM; Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone) and M-CSF (5 ng/mL) for 12 h. The non-adherent cells were harvested and were further cultured for 3 days with α-MEM containing M-CSF (30 ng/mL) to generate osteoclast precursors. Osteoclast differentiation was induced by incubating osteoclast precursors in the presence of M-CSF (30 ng/mL) and RANKL (100 ng/mL) with or without the aqueous suspensions of P. coreanum powder for 4 days. TRAP staining was performed with the use of a leukocyte acid phosphatase staining kit (Sigma-Aldrich), following the manufacturer’s instructions. TRAP-positive multinucleated cells having more than three or ten nuclei were counted using a light microscope. For measuring the size of TRAP-positive multinucleated osteoclasts containing more than ten nuclei, 90 multinucleated cells from four or more random fields were traced using ImageJ software (NIH, Bethesda, MD, USA).

Osteoclasts were differentiated from bone marrow cells of XBP‐1s^+/+pink1^−/− and XBP‐1s^+/+pink1^+/+ mice (8–12 weeks old). Cells were differentiated with 40 ng mL⁻¹ of mouse macrophage colony‐stimulating factor (M‐CSF, R&D Systems) in α‐MEM containing 10% FBS for 3 days, and additional 100 ng mL⁻¹ of mouse RANKL (Sigma‐Aldrich) was added to continue culture for additional 3 days. Tartrate‐resistant acid phosphatase (TRAP) staining of osteoclasts was performed using a leukocyte acid phosphatase staining kit (Sigma‐Aldrich).

Tetrandrine (purity >98%) was purchased from MedChemExpress (New Jersey, USA) and was dissolved in DMSO at a 10 mmol/L stock solution and stored -20℃. Further dilution was carried out in culture medium for cells and PBS medium for animals. Primary antibodies against CTSK, CTR, MMP-9, TRAF6, TRAP, GAPDH, and β-actin were purchased from Proteintech (Wuhan, Hubei, China). Primary antibodies against NFATc1, P-PI3K, AKT, P-AKT, P50, P-P50, P65, P-P65, IκBα, P-IκBα, ERK1/2, P-ERK1/2, JNK, P-JNK, P38, and P-P38 were obtained from Cell Signaling Technologies (Beverly, MA, USA). Primary antibodies against RANKL, OPG, and the ELISA kit of RANKL were obtained from ABclonal (Wuhan, Hubei, China). The ELISA kits of OPG, IL-6, TNF-α, TRAcp5B, and CTX-I were purchased from Sangon (Shanghai, China). A CCK-8 assay kit was purchased from Dojindo (Tokyo, Japan). A leukocyte acid phosphatase staining kit was obtained from Sigma‐Aldrich (MO, USA). Recombinant M‐CSF and Recombinant m-RANKL were obtained from R&D Systems (Minneapolis, MN, USA). The cell culture medium that alpha‐modified minimal essential medium (α‐MEM), fetal bovine serum (FBS), and penicillin-streptomycin were purchased from Thermo Fisher Scientific (Scoresby, Vic., Australia).

For the osteoclast differentiation assay, mouse bone marrow macrophages (BMMs) were harvested from bone marrow cells in the long bones of 8-week-old wild-type mice (C57BL/6J, The Jackson Laboratory, Sacramento, CA, USA). In brief, bone marrow cells were flushed out from the femurs and tibias, treated with red blood cell lysis buffer, and suspended in 10% fetal calf serum (FCS) and 1% penicillin/streptomycin (Corning, New York, NY, USA). Cells were cultured in the presence of M-CSF (10 ng/mL, R&D Systems, Minneapolis, MN, USA, 416-ML), and 1 day later, non-adherent cells were plated at a density of 0.5 × 10⁶ cells/well in 24-well plates. BMMs were differentiated into osteoclasts by treating them with M-CSF (10 ng/mL) and RANKL (20 ng/mL, R&D Systems, 462-TEC), and two days later, liquid chlorobenzene was added daily at four concentrations (0 (control), 1, 10, and 100 µg/mL) for 14 days. To assess osteoclast differentiation, TRAP staining was performed using a leukocyte acid phosphatase staining kit (Sigma, 387A) according to the manufacturer’s protocol. The TRAP-stained osteoclasts were detected using an Evos microscope (Applied Biosystems, Waltham, MA, USA).