M csf 416 ml

Pan-caspase inhibitor, z-VAD-fmk (#A1902) was obtained from ApexBio (Houston, USA). Lactacystin (L6785), MG132 (C2211), cyclohexamide (C4859), hydroxychloroquine (H0915), and ultrapure LPS (E. coli 0111:B4, L3024) were obtained from Sigma-Aldrich (Oakville, Canada). Necroatatin-1 (9037) was obtained from Sigma Chem Co. (Oakville, Canada). M-CSF (416-ML) and TNFα (410-MT) were obtained from R&D (Minneapolis, USA). Birinapant (S7015) was obtained from Selleckchem (Houston, USA). GSK843 and GSK'872 (AOB4898, AOB488) were obtained from Aobious (Gloucester, USA). Caspase-8 Inhibitor, z-IETD-FMK (064-20C) was obtained from BioVision (San Francisco, USA). P38 inhibitor (5633S) was obtained from Cell Signaling (Danvers, USA). Alkaline Phosphatase, Calf Intestinal (CIP) (M0290, NEB). Protease inhibitors (04693132001) were obtained from Roche Applied Science (Laval, Canada). PR-619 (SI9619), 1,10-phenanthroline (SI9649) and GST-tagged tandem ubiquitin-binding entities (TUBE) (UM102) were obtained from LifeSensors (Malvern, USA). Actinomycin-D (0219452505) was obtained from MP Biomedicals (Solon, USA). Necrostatin-1s (cat# 504297) and MG341 (Bortezomib) (cat# 504314) were obtained from Calbiochem (San Diego, CA, USA). Zombie Yellow™ Fixable Viability Kit cat# 423103, Biolegend (San Diego, USA).

Methoxy terminated PLGA-PEG (10:5 kDa) was purchased from Polyscitech (West Lafayette, IN). Polyvinyl alcohol (PVA, 25 kDa) was purchased from Polysciences (Warrington, PA). Antibody to L-plastin (SC-16657) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Phosphoserine (125277) antibody was bought from Abcam (Cambridge, MA). Recombinant TNF-α (216-TA) and MCSF (416-ML) were purchased from R&D Systems (Minneapolis, MN). Protein estimation reagent, molecular weight standards for proteins, and PAGE reagents were bought from Bio-Rad. HRP-conjugated secondary antibodies for immunoblotting were obtained from GE Healthcare. PLGA (7-17 kDa, 50:50), dichloromethane (DCM), anti-rabbit antibodies to GAPDH, Rhodamine-phalloidin, and other chemicals were purchased from Sigma-Aldrich (St. Louis, MO). sNT-LPL peptides (fluorescence-conjugated and unconjugated) are made from Genscript Co. (Piscataway (NJ)).

WT or PIP5k1β^−/− BMMs were obtained as described previously (Qin et al., 2012 (link); Li et al., 2013 (link)). In brief, bone marrow cells extracted from the femurs and tibiae of 9–10-week-old WT and littermate PIP5k1β^−/− mice were cultured in α-MEM medium containing 15 ng/ml M-CSF (416-ML; R&D) in a T-75 cm² flask for proliferation for 2–3 days until reaching 90% confluence. The cells were washed with phosphate buffer solution (PBS) three times and trypsinized for 30 min. Adherent cells were classified as BMMs. WT and PIP5k1β^−/− BMMs were plated on 96-well plates at a density of 8 × 10³ cells/well in triplicate and incubated with α-MEM medium containing 20 ng/ml M-CSF in a humidified incubator containing 5% CO₂ at 37°C for 2 days. The cells were then cultured with α-MEM medium with M-CSF (20 ng/ml), RANKL (75 ng /ml; 315-11; Peprotech), and indicated inhibitors for indicated times. The medium was changed every other day. The inhibitors for p38/MAPK (SB203580), MAPK/ERK (U0126-EtOH), and JNK (SP600125) were purchased from Selleckchem. For osteoblast differentiation, BMSCs were gained from 9–10-week-old mice and expanded as previously described, induced by culturing cells in osteogenic medium (DMEM containing 1 M β-glycerophosphate, 50 mM ascorbic acid, and 1 mM methylisobutylxanthine) for indicated times, followed by subsequent experiments.

Bone marrow from tibia and femur of WT and cKO mice was flushed with α-MEM and cells pelleted by centrifugation at 1500 rpm for 5 min. Bone marrow cells were then cultured in complete α-MEM media supplemented with 10% fetal bovine serum, 1% antibiotic, and 30 ng/ml of recombinant mouse macrophage colony stimulating factor (MCSF, 416-ML, R&D systems, Minneapolis, MN, USA) at 37 °C in a humidified 5% CO₂ atmosphere to allow cell attachment. After 2 days, non-adherent cells were removed and discarded; adherent cells were cultured to confluence and treated as bone marrow macrophages (BMMs). BMMs of passage 1 or 2 were used in the experiments below. BMMs were seeded on to 96-well plate (6 × 10³ cells/well) and treated with 30 ng/ml MCSF, and 100/25 ng/ml RANKL (462-TEC, R&D systems) to induce OC formation. The media was replaced with supplemented MCSF and RANKL (with and without BS) every 2 days and after 7 days of culture, the cells were fixed in 2.5% glutaraldehyde and stained with Acid Phosphatase staining kit (387 A, Sigma–Aldrich, St. Louis, MO, USA) according to the protocol of the manufacturer. TRAP-positive multinucleated cells with more than three nuclei were counted as OCs using light microscopy. The number of OCs in each well was counted using ImageJ software.