Osteoblast-Induced Osteoclast Apoptosis by FAS Ligand/FAS Pathway is
Required for Maintenance of Bone Mass
Lei Wang, Shiyu Liu, Yinghua Zhao, Dawei Liu, Yi Liu, Chider Chen, Saoussen Karray, Songtao Shi,
Yan Jin
SUPPLEMENTARY EXPERIMENTAL PROCEDURES
Antibodies. Anti-mouse TNF-α, FASL, FAS and RANK antibodies were purchased from Santa Cruz
Biotech (Santa Cruz, CA); IFN-γ antibody was purchased from Abbiotec (San Diego, CA);
phosphorylated nuclear factor κB (p-NFκB), NFκBp65, phosphorylated nuclear factor κ B inhibitor
(p-IκB) and IκBα were purchased from Cell Signaling (Danvers, MA). Anti-β-actin antibody was
purchased from Sigma-Aldrich (St. Louis, MO).
Skeletal analyses and radiographic examinations. We euthanized the newborn mice and fixed the
whole skeleton in 4% paraformaldehyde (PFA) for 18 h. We cleared and stained skeletons with Alcian
blue for cartilage and Alizarin red for bone. For radiographic examinations, we analyzed the skeletons
by contact radiography with a Digital X-ray Specimen PRO System (Carestream Health, Inc., Upland,
CA).
Isolation of mouse bone marrow osteoblast progenitors. For isolation of bone marrow osteoblast
progenitors, bone marrow cells were flushed out from bone cavity of femurs and tibias of mice with
2% heat-inactivated fetal bovine serum (FBS; Equitech-Bio, Kerrville, TX) in PBS. A single-cell
suspen- sion of all nucleated cells was obtained by passing all bone marrow cells through a 70-μm cell
strainer (BD Bioscience, San Jose, CA), respectively. All the single cells were seeded into 100-mm
culture dishes (Corning, Corning, NY) and initially incubated for 48 h at 37 °C and 5% CO 2. To
eliminate the nonadherent cells, the cultures were washed with PBS twice on the second day. The
attached cells were cultured for 16 d with alpha minimum essential medium (α-MEM, Invitrogen,
Grand Island, NY) supplemented with 20% FBS, 2 mM L-glutamine (Invitrogen), 55 μM
2-mercaptoethanol (Invitrogen), 100 U/ml penicillin and 100 μg/ml streptomycin (Invitrogen). To
confirm mesenchymal stem cell character, flow cytometric analysis was used to show that these
osteoblast progenitors were positive for CD73, CD90, SCA-1 and SSEA-4 and negative for CD11b,
CD31, CD34 and CD45.
Population-doubling and proliferation assays. Population doublings (PD) for each passage was
determined by using the formula PD≈log2[Nc/No], where No is the original cell population and Nc is
the number of cells at confluence, and by adding the PD from each passage together to obtain the PD
values. The PD assay for mouse osteoblast progenitors began with passage 1 (P1). For proliferation
assay, P1 cells (1 × 104 per well) were seeded on 2-well chamber slides (Nunc) for 1–2 days, incubated
with BrdU reagent (1:100, Invitrogen) for 24 hrs, and stained with the BrdU staining kit (Invitrogen),
following the manufacturer’s instructions. The cells were lightly stained with hematoxylin solution
(Invitrogen). To quantify cell proliferation capacity, we used 10 representative images to calculate
BrdU-positive nuclei numbers. Cell proliferation was shown as a percentage of BrdU-positive nuclei
over total nucleated cells. Adipogenic differentiation assay. For the adipogenic induction assay,
osteoblast progenitors were cultured to confluence and then induced under an adipogenic medium
containing 500 μM isobutyl-methylxanthine, 60 μM indomethacin, 0.5 μM hydrocortisone, and 10 μM
insulin for 1 week. Cultures were stained with 0.3% Oil red-O after adipogenic induction. The Oil-red
O-positive droplets-containing cells were counted and shown as a percentage of Oil-red O-positive
cells over total cells. Western blot analysis. Total protein was extracted using M-PER mammalian
protein extraction reagent (Thermo). Nuclear protein was obtained using NE-PER nuclear and
cytoplasmic extraction reagent (Thermo). Protein was applied and separated on 4-12% NuPAGE gel
(Invitrogen) and transferred to ImmobilonTM-P membranes (EMD Millipore, Billerica, MA). After
blocking with 5% non-fat dry milk for 1 hour, the membranes were incubated with the primary
antibodies (1:200-1000 dilution) at 4 ºC overnight. Horseradish peroxidase-conjugated IgG (Santa
Cruz) was used to treat the membranes for 1 hour and subsequently treated with a chemiluminescent
substrate (Thermo). The bands were detected on BIOMAX MR films (Kodak). Each membrane was
also stripped using a stripping buffer (Thermo) and reprobed with anti-β-actin to determine the loading
amount. Flow Cytometry Analysis for HSCs and T cell subsets. Cells were stained by standard
protocols with the following antibodies (eBiosciences unless otherwise noted). To determine the
population of HSC-enriched Lineage–Sca-1+c-Kit+ (LSK) cells, long-term HSCs (LT-HSCs, Lineage–
Sca-1+c-Kit+Flk2–CD34–) and short-term HSCs (ST-HSCs, Lineage–Sca-1+c-Kit+Flk2–CD34+), bone
marrow cells freshly harvested from femurs and tibias were stained with a cocktail of PE-conjugated
B220, TER119, CD3e and CD41; PerCP-conjugated Sca-1; APC eFluor780-conjugated c-Kit;
FITC-conjugated CD34 and APC-conjugated Flk2. Multiparameter FACS analysis was performed on
a LSRII (BD Bioscience, San Jose, CA). For analysis of Th1 and Th2 cells, 1–2×106 splenocytes were
activated with 5 ng ml-1 phorbol 12-myristate 13-acetate (PMA, Sigma Chemical Co., St. Louis, MO),
250 ng ml-1 ionomycin (Sigma) and 1 μg ml-1 brefeldin A (GolgiPlug, BD Bioscience, San Jose, CA)
for 4 hours at 37°C and 5% CO2, followed by incubation with 1 μg of PerCP-conjugated anti-CD4
antibody for 30 min on ice under dark condition. After cell fixation and permeabilization, cells were
stained with 1 μg of APC-conjugated anti-IFN-γ or IL-4. Isotypematched APC- or PerCP- conjugated
IgGs were used as controls. For Foxp3 intercellular staining, T cells were stained with anti-CD4 and
-CD25 antibodies (1 mg each) for 30 min on ice. Next, cells were stained with anti-Foxp3 antibody
using Foxp3 staining buffer kit (eBioscience). For IL17 staining, T cells were stained with anti-CD4
antibody and then stained with anti-IL17 antibody using Foxp3 staining buffer kit. After washing with
PBS/0.4%BSA 3 times, all samples were analyzed with FACScalibur (BD Bioscience, San Jose, CA).
Apoptosis PCR array. To profile the expression of key genes involved in programmed cell death, a
mouse Apoptosis RT² Profiler PCR Array (SABiosciences, Frederick, MD) was used according to the
protocol of the manufacturer. Briefly, total RNA was isolated from the cultures using SV total RNA
isolation kit (Promega, Madison, WI) according to manufacturer’s instructions. The cDNA was
synthesized from 100 ng of total RNA using RT² First Strand Kit (SABiosciences). cDNA was added
to SYBR Green qPCR Master Mixes (SABiosciences), and the mixture was aliquoted across the PCR
Array, followed by running in the BioRad CFX96TM Detection System (BioRad). Data analysis was
performed
on
the
manufacturer’s
Web
portal
at
http://www.SABiosciences.com/pcrarraydataanalysis.php by converting raw Ct data to fold–change
results.