Human primary skin fibroblasts and HeLa cells were grown in DMEM; MC3T3-E1, OB6, and primary cells from murine calvariae, spleen, or bone marrow were grown in α-MEM. All media contained 4500 mg/L glucose and 110 mg/mL sodium pyruvate (HyClone, Thermo Fisher Scientific, Pittsburgh, PA, USA) and were supplemented with 10% fetal bovine serum (FBS), L-glutamine (2 mM), 100 units/mL penicillin, and 100 μg/mL streptomycin. Ascorbic acid (100 µg/mL) and β-glycerophosphate (5 mM) were added to the cell culture medium as indicated. Primary osteoblasts from calvaria were obtained using standard procedures as we have described. To induce ER stress, osteoblasts were treated with 2 µg/mL tunicamycin for 0, 8, 12, 16, and 20 hours. Whole bone marrow cultures were established by harvesting femurs and tibias, flushing out the marrow with a 25-gauge needle under sterile conditions, filtering the marrow through a 70-μm mesh, and removing red blood cells with a commercially available solution (Miltenyi Biotec, Auburn, CA, USA). Bone marrow was plated at the density of 2 × 106 cells/well in a 24-well plate in presence of 1α,25 dihydroxyvitamin D3 (10−8M) (Sigma Aldrich, St. Louis, MO, USA; cat. # D1530). Similarly, splenocyte cultures were established by harvesting spleens, disaggregating the tissue, and filtering the cells through a 70-µm mesh. Splenocytes (without red blood cells) were seeded on either plastic or Osteo Assay (Corning Inc., Corning, NY, USA) 96-well plates at 1.75 × 105 cells/well in presence of macrophage colony-stimulating factor (M-CSF) and RANKL (100 ng/mL) and stained for tartrate-resistant acid phophatase (TRAP) activity after 5 to 7 days. Bone marrow macrophage cultures were prepared from cell culture plastic nonadherent bone marrow cells, which were expanded for 24 to 48 hours with M-CSF and then harvested, counted, and seeded on either plastic or dentine (Immunodiagnostic Systems [IDS], Scottsdale, AZ, USA) in 96- or 48-well plates at 1 × 106 cells/well with M-CSF and RANKL (100 ng/mL). Both M-CSF and RANKL used in all osteoclast assays were kindly provided by Dr Bryant G Darnay as conditioned medium from L929 cells (used at 5%) and as His-RANKL purified recombinant protein, respectively. Cells were removed from the dentine slices with fresh 20% bleach solution in PBS for 30 minutes. Bone resorption pit area was quantified using Osteomeasure software (Osteometrics, Atlanta, GA, USA) after the staining with 1:20 wheat germ agglutinin (WGA)-lectin conjugated to TRITC (Sigma-Aldrich, St. Louis, MO, USA) for 1 hour. Mouse Sc65 cDNA subcloned in pcDNA3.1 myc/His A (Invitrogen, Grand Island, NY, USA) was sequenced to confirm its identity and then transfected into HeLa cells using X-tremeGENE according to the manufacturer's instructions (Roche Applied Science, Indianapolis, IN, USA). Mission Lentiviral Transduction particles encoding shRNA against Sc65 or TurboGFP were purchased from Sigma-Aldrich and used according to the recommended transduction protocol from the manufacturer. OB6 cells were plated in a 12-well plate at two different densities (0.5 and 0.25 × 105 cells/well) and the next day transduced with lentiviral particles in the presence of 8 μg/mL hexadimethrine bromide at an MOI of 5. Selection with puromycin (2 μg/mL) began on the second day after transduction, and drug-resistant OB6 cells were always maintained under puromycin selection.