Recombinant rankl

Bone marrow (BM) cells were isolated from 8-week-old male C57BL/6J mice as described previously [21 (link)]. Mice were bred and housed at the animal housing unit (College of Science, King Faisal University, Saudi Arabia) under standard conditions (21°C, 55% relative humidity) on a 12-hour light/12-hour dark cycle, and ad libitum food (Altromin® Spezialfutter GmbH & Co., Germany) and water were provided in accordance with the ethical clearance of the Standing Committee on Research Ethics. Mice were sacrificed by cervical dislocation and BM was flushed out from tibia and femur. BM was centrifuged for 1 min at 400 g and filtrated through a 70 μm nylon mesh filter. BM cells were then plated in 96‐well plates at a density of 1 × 10⁶ cells/well in osteoclast differentiation medium (ODM) containing α‐MEM (Gibco BRL, Carlsbad, CA, USA) supplemented with 10% FBS (Gibco BRL), 100 U/mL of penicillin (Gibco BRL), 100 μg/mL of streptomycin (Gibco BRL), 25 ng/mL of recombinant M‐CSF (R&D Systems, Minneapolis, MN, USA), and 25 ng/mL of recombinant RANKL (Pepro‐Tech, Rocky Hill, NJ, USA) to induce osteoclast formation. Cells were maintained at 37°C in a 5% CO₂ incubator, and the medium was changed every 3 days.

Thymic lobes were isolated from E15.5 WT and Jmjd6^−/− embryos, and were cultured for 4 days on Nucleopore filters (Whatman) placed in RPMI 1640 medium (Life Technologies) supplemented with 10% heat-inactivated fetal calf serum (FCS) (Nichirei Bioscience), 50 μM 2-mercaptoethanol (Nacalai tesque), 2 mM L-glutamine (Life Technologies), 100 U ml^–1 penicillin (Life Technologies), 100 μg ml^–1 streptomycin (Life Technologies), 1 mM sodium pyruvate (Life Technologies), MEM non-essential amino acids (Life Technologies) and 1.35 mM 2-DG (Sigma-Aldrich). After cultivation in complete RPMI medium without 2-DG for one more day, four pieces of WT and Jmjd6^−/− fetal thymi were grafted under the renal capsule of athymic (nude) and WT C57BL/6 mice. Thymic chimeras were analysed 6–10 weeks after transplantation. In some experiments, TECs were stimulated in vitro with recombinant RANKL (1 μg ml^–1; Peprotech), agonistic anti-LtβR antibody 3C8 (2 μg ml^–1; eBioscience) and/or recombinant CD40L (5 μg ml^–1; R&D systems) for 4 days before analyses.

BMMs were isolated and cultured as previously described (32 (link)). Briefly, both ends of the femur and the tibia were removed before the bone marrow was flushed out with cell culture media. Bone marrow cells were cultured for 3 days on Petri dishes in growth media; the growth media were reconstituted from a custom-made MEMα powder (Gibco) with addition of fresh glucose, pyruvate, and glutamine, supplemented with 10% FBS (Atlanta Biologicals) together with 10% CMG14-12 cell culture supernatant. The CMG14-12 supernatant containing M-CSF was prepared as previously described (33 (link)). The adherent BMMs were then dissociated with 0.25% Trypsin-EDTA (Gibco) and reseeded with growth media in 96- or 24-well plates at the density of 7.5 × 10⁴ cells/cm². On the following day, the growth media were replaced with differentiation media, i.e., reconstituted custom-made MEMα containing 10% FBS, 1:50 CMG14-12 supernatant, and 75 ng/mL recombinant RANKL (PeproTech). Osteoclasts were detected with TRAP staining (MilliporeSigma).

Bone marrow cells derived from mice were seeded (2.5 × 10⁵ cells per well in a 24 well plate) and cultured in α-minimum essential medium (α-MEM) with 10% fetal bovine serum (FBS) containing 10 ng/mL recombinant M-CSF (PeproTech). After two days, adherent cells were used as BMMs. These osteoclast progenitor cells were further cultured in the presence of 50 ng/mL recombinant RANKL (PeproTech) and 10 ng/mL recombinant M-CSF to generate mature osteoclasts for 48–72 h. The presence of osteoclasts was confirmed by TRAP staining. TRAP-positive multinuclear cells (more than three nuclei) were counted.

Recombinant RANKL and M-CSF were purchased from PeproTech (Princeton, NJ, United States). Icariside I (ICS; C₂₇H₃₀O₁₁; MW: 530.53), baohuoside Ⅰ (BS; C₂₇H₃₀O₁₀; MW: 512.52), icariin (ICA; C₃₃H₄₀O₁₅; MW: 676.68), icaritin (ICT; C₂₁H₂₀O₆; MW: 368.38), and a Cell Counting Kit-8 (CCK-8) were purchased from Target Molecule Corp. (Boston, MA, United States). Trypsin-EDTA (0.05%), PBS, FBS, and MEM-Alpha basic were purchased from Gibco. Antibodies against p38 (#8690), p-p38 (#4511), ERK (#9102), p-ERK (#4370), JNK (#9252), p-JNK (#4668), p65 (#8242), p-p65 (#3033), IκBα (#4812), p-IκBα (#2859), and RANK (#4845) were purchased from Cell Signaling Technology (Boston, MA, United States). Antibodies against TRAP (ab52750) and cathepsin K (ab19027) were purchased from Abcam (Cambridge, MA, United States). Antibodies against NFATc1 (66963-1-Ig), uPAR (10286-1-AP), and β-actin (66009-1-Ig) were purchased from Proteintech Group Inc. (Wuhan, China). DAPI and Actin-Tracker Green were obtained from Beyotime (Shanghai, China). Cy3-labelled goat anti-rabbit antibody was purchased from Boster (Wuhan, China). Unless noted otherwise, other reagents were of the highest purity available and were obtained from Sigma-Aldrich (St. Louis, MO, United States).

Rapamycin (R117), Bafilomycin A1 (B1793), and 3-MA (M9281) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s media (DMEM): nutrient mixture F-12 (12400–024) and fetal calf serum (FCS) (10099–141) were obtained from Gibco (Grand Island, NY, USA). RIPA lysis buffer (P0013C) and protease inhibitor cocktail for general use (P1005) were acquired from Beyotime (Haimen, China). Recombinant M-CSF (400–23) and Recombinant RANKL (400–30) were obtained from PeproTech (Rocky Hill, NJ, USA). beclin1-shRNA (LV-beclin1-shRNA), beclin1 (LV-beclin1), and control (LV-control) lentiviral vector constructs were generated and produced by GenePharma (Shanghai, China). Polyclonal antibodies against beta-actin (β-actin), CD68, beclin1, LC3II/I, LC3B-II, ATG5, P62, RANKL, OPG, and Vimentin were purchased from Novus Biologicals (Littleton, CO, USA).