Bone resorption assay kit

The activity of differentiated osteoclast was performed by the bone resorption assay kit (Cosmo Bio. Co. Ltd., Japan) according to the manufacturer's instructions. BM cells were cultured on fluoresceinated calcium phosphate-coated 24-well plates in the presence of M-CSF and RANKL without or with 5′-HA for 6 days. Fluorescent released from the calcium phosphate layer into conditioned medium due to osteoclast resorption activity was measured by detecting the fluorescence intensity at an emission wavelength of 535 nm.

PPOA-N-Ac-2-Cl, purchased from ChemBridge (San Diego, CA, USA), was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA) to obtain a 50-mM solution. Aliquots of the solution were stored at −20 °C and diluted to the appropriate concentrations in cell culture medium immediately before use. DMSO was used as the vehicle control. Alpha-modified minimal essential medium (α-MEM) and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Bone resorption assay kit was procured from Cosmo Bio Co., Ltd., Tokyo, Japan. BCA protein assay kit was purchased from Pierce Biotechnology, (Rockford, IL, USA). Primary antibodies including anti-β-actin (Sigma-Aldrich, St Louis, MO, USA), anti-c-Src (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-cathepsin K (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-ERK1/2, anti-phospho-ERK1/2, anti-AKT, anti-phospho-AKT, anti-IκBa, anti-phospho-IκBa, anti-p38, anti-phospho-p38, anti-JNK, anti-phospho-JNK, anti-c-fos, anti-NFATc1, anti-p65, and anti-phospho-p65, anti-TRAF6, anti-calcineurinA, anti-calmodulin were purchased from Cell Signaling Technology (Boston, MA, USA). Horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from Cell Signaling Technology, and chemiluminescence signals were detected using an ECL system (iNtRON, Seoul, Korea).

We performed an in vitro resorption pit assay using a bone-resorption assay kit (Cosmo Bio Co., Ltd., Tokyo, Japan). Monocytes were cultured on a bone-coating plate with M-CSF in the presence or absence of various concentrations of IL-26 (1, 50, and 100 ng/mL) for 14 days. The cells were removed from the bone-coating plate by wiping the surface, and the numbers of pits formed by bone resorption on the plate were counted.

Bone resorption activity was determined using bone resorption assay kit (CosMo Bio, Tokyo, Japan), as described previously [45 (link)]. Briefly, BMMs were cultured on a calcium phosphate-coated 48-well plate (2.5 × 10⁴ cells/well) in the presence of M-CSF (30 ng/mL) and RANKL (100 ng/mL) with different concentrations of PSTP-3,5-Me (0, 3, and 10 µM) for six days. The fluorescence intensity of the culture supernatant was analyzed using a fluorescence plate reader, SpectraMax i3x (Molecular Devices, San Jose, CA, USA) with 485 nm and 535 nm wavelengths. The pit area was calculated using Image J software.

Bone resorption was assessed by the bone resorption assay kit (Cosmo Bio Co., Ltd., Tokyo, Japan), as previously described [45 (link)]. The BMMs were seeded into a 48-well bone resorption assay plate at a density of 2.5 × 10⁴ cells/well. The cells were differentiated with M-CSF and RANKL in the absence or presence of indicated concentrations of LSD1 inhibitors. After cell differentiation, the attached cells were removed using 5% sodium hypochlorite. The plates were air-dry at RT and resorption pits were captured by a microscope. In addition, the resorption areas were quantified by analyzing three randomly selected pictures per well by Image J software.

Neocuproine, Dulbecco’s modified Eagle’s medium (DMEM), minimum essential medium alpha medium (α-MEM), fetal bovine serum (FBS), 2,5-diphenyltetrazolium (MTT), Triton X-100, RANKL, sodium tartrate, p-nitro-phenylphosphate (PNPP), fluoresceinamine-labeled chondroitin sulfate (FACS), phosphate-buffered saline (PBS, pH 7.4), ethylenediaminetetraacetic acid (EDTA), dithiothreitol (DTT), phenylmethanesulfonyl fluoride (PMSF), dimethylsulfoxide (DMSO), chlorogenic acid, caffeic acid, isoquercitrin, and isochlorogenic acid A were purchased from Sigma-Aldrich (St. Louis, MO, USA). Hyeroside was purchased from Hwz Analytic GmbH (Ruelzheim, Germany). Scoparone was obtained from Phytolab GmbH&Co (Vestenbergreuth, Germany). Antibodies and protein A-agarose beads were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), including anti-TRAF6 (sc-7221), V-ATPase (sc-20946), and β-actin (sc-130656). A bone resorption assay kit was purchased from CosMo Bio Co., Ltd. (Tokyo, Japan). MC3T3-E1 subclone 4 and RAW 264.7 cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA).