Supplementary Material and Methods
Immunofluorescence staining
BMMs were seeded at a density of 1 × 104 cells/well in a 96-well black clear plate (Corning, NY) and incubated with RANKL (10 ng/ml) and M-CSF (30 ng/ml) for 3 days to generate preosteoclasts. Then, preosteoclasts were pretreated with each polyamine (putrescine, 100 μM; spermidine, 10 μM; putrescine, 10 μM) or a selective Ca2+ chelator, BAPTA-AM (Sigma, MO; 2 μM) in the presence of M-CSF (30 ng/ml) for 2 hours. And then, cells were incubated with RANKL (10 ng/ml) for 15 minutes, fixed with 3.7% formaldehyde, permeabilized with 0.1% Triton X-100 for 10 min, and then incubated with anti-NFATc1 (Santa Cruz Biotechnology, CA) for 2 hours. Cells were washed with PBS contain 0.1% BSA and incubated with Alexa Flour 633-conjugated secondary antibody for 1 hour, and then incubated with 10 mg/ml Hoechst33342 (Carlsbad, CA). Images and densities of stained cells were analyzed by Cellomics arrayscan (Thermo Scientific). Fluorescent intensity of nuclear NFATc1 was normalized to DNA content (or the total nuclear intensity of Hoechst33342) in each individual cell.
Intracellular Ca2+ measurement
BMMs were seeded at a density of 1 × 104 cells/well in a 96-well black clear plate (Corning, NY) and incubated with RANKL (10 ng/ml) and M-CSF (30 ng/ml) for 3 days to generate preosteoclasts. Then, preosteoclasts were incubated with Fura-2,AM (Life technology, CA; 5 μM) and Pluronic F-127 (Sigma, MO; 0.05%) for 30 minutes, washed twice with the media, and incubated with HBSS (Hank’s Balanced Salt Solution; gibco Gaithersburg, MD). Then, intracellular Ca2+ levels were measured by BD Pathway Bioimaging Systems. Fluorescent dyes inside cells were excited at wavelengths of 340 nm and 380 nm sequentially, and the emitted fluorescence was passed through 510 nm cut-off filter. Each image was collected with a CCD camera and analyzed by AttoVision software. All results were digitized to the mean of the ratio (340 nm/380 nm). For a total duration of 300 seconds, RANKL (10 ng/ml) or BAPTA-AM (2 μM) was added into cells 150 seconds after the start.
NF-κB luciferase activity assay
Luciferase reporter plasmid, NF-κB-luc reportor vector, was purchased from Clontech (CA). The full-length human RANK cDNA was amplified from human leukocyte cDNA from Clontech (CA) and cloned into the HindIII-EcoRI site of pcDNA3.1 (Invitrogen). Recombinant human RANKL (hRANKL) was purchased from R&D Systems (CA). For measuring the luciferase activity, human embryonic kidney HEK293T cells were plated at a density of 5 × 104 cells/well on a 48-well plate in triplicate for 2 days. NF-κB-luc (100 ng/well) and pcDNA3.1-RANK (100 ng/well) were transfected into HEK293T cells with Lipofectamine 2000 (Invitrogen, CA) according to the manufacturer’s protocol. After transfection for 12 hours, HEK293T cells were cultured for an additional 1 day. After each wells was pretreated with NF-κB activation inhibitor (CALBIOCHEM, cat. no. 481406) or each polyamin for 2 hours, and incubated with hRANKL (50 ng/well) for 1 day. The luciferase activity was detected in cell extracts with the Dual-Luciferase Reporter Assay System (Promega, WI) and Wallac EnVision microplate reader (PerkinElmer). Luciferase activity was normalized to the amount of proteins used in each well, and represented to the relative fold induction.
Statistical analysis
All quantitative values were presented as mean ± SD. Statistical differences were analyzed using Student’s t-test. A value of p < 0.05 was considered significant.
Supplementary Figure legends
Figure S1. Effect of polyamines on RANKL-induced NF-κB activation. The effects of polyamines or NF-κB activation inhibitor on the RANKL-induced transcriptional activity of NF-κB were evaluated by luciferase activity assay. ##, P < 0.01 (vs. the control); *, P < 0.05; ** P < 0.01 (vs. the group treated with hRANKL alone)
Figure S2. Effect of polyamines on RANKL-induced nuclear translocation of NFATc1 in preosteoclasts. (A) Preosteoclasts derived from BMMs cultured with M-CSF (30 ng/ml) and RANKL (10 ng/ml) for 3 days were pretreated with each polyamine (putrescine, 100 μM; spermidine, 10 μM; putrescine, 10 μM) or 2 μM BAPTA-AM, a selective Ca2+ chelator in the presence of M-CSF (30 ng/ml) for 2 hours. And then, cells were incubated with RANKL (10 ng/ml) for 15 minutes and the immunofluorescence staining was carried out for visualizing the location of NFATc1. Images (A) and densities (B) of NFATc1-stained cells were analyzed by Cellomics arrayscan (Thermo Scientific). ##, P < 0.01 (vs. the controls); **, P < 0.01; *** P < 0.001 (vs. the RANKL-treated group)
Figure S3. Effect of RANKL or BAPTA-AM on oscillation of intracellular Ca2+ in preosteoclasts. In reosteoclasts, the effect of RANKL (10 ng/ml; A) or BAPTA-AM (2 μM; B) on the oscillation of intracellular Ca2+ was evaluated by analyzing the fluorescent dye-based images of cells (n=6) in BD Pathway Bioimaging Systems
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