1
Supporting Information
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3
Hoechst staining
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BMMs (1  104 cells/well) were seeded in a 96-well plate, treated with the vehicle
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(0.1% DMSO) or praeruptorin A (10 μM) for 2 h in the presence of M-CSF (30 ng/ml),
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and incubated with RANKL (10 ng/ml) for 1 and 3 days. Then, cells were fixed with
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10% formalin for 5 min, permeabilized with 0.1% Triton X-100 in PBS for 5 min,
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washed twice with PBS, and incubated with 10 μg/ml Hoechst 33342 (Carlsbad, CA)
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for 5 min. Images of stained nucleus were captured under a fluorescent microscope with
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a DP Controller.
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Osteoclast differentiation
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To evaluate the stage-dependent anti-osteoclastogenic activity of praeruptorin A, BMMs
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were isolated and cultured with praeruptorin A (10 M) for various times periods in the
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presence of M-CSF and RANKL for osteoclast differentiation. After TRAP staining,
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TRAP-positive multinuclear cells with three or more nuclei were counted as osteoclasts.
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Furthermore, in order to investigate whether age, strain or sex could affect anti-
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osteoclastogenic activity of praeruptorin A, mice (male and female ICR strain, 5-week
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old; male and female C57BL6/N strain, 5-week old and 8-month old) were purchased
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from Central Lab. Animal Inc. (Seoul, Korea), and as described in the ‘Materials and
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Methods’, BMMs were isolated and cultured with praeruptorin A in the presence of M-
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CSF and RANKL for osteoclast differentiation.
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Actin ring staining
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On the differentiation day 4, cells were fixed with 10% formalin for 5 min,
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permeabilized with 0.1% Triton X-100 in PBS for 5 min, and washed with PBS. Actin
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rings were stained with 50 μg/ml phalloidin-FITC (Sigma-Aldrich, MO) for 30 min
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under the condition of light protection. After washing twice with PBS, nuclei were
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stained with 10 μg/ml Hoechst 33342 for 5 min. Images of stained nucleus were
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captured under a fluorescent microscope with a DP Controller.
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Bone pit formation analysis
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BMMs and osteoblasts isolated from the calvariae of newborn mice by serial digestion
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with collagenase were co-cultured with 1α, 25(OH)2D3 (10-8 M) and PGE2 (10-6 M) in
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the collagen-coated dishes. After 7 days, multinucleated osteoclasts were replated on
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BioCoat Osteologic MultiTest slides and after 2 h incubation, cells were further
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incubated with praeruptorin A and RANKL for 6 h. Then, cells were stained for TRAP,
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and photographs were taken under a light microscope at 40  magnification. To observe
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resorption pits, cells on the BioCoat slides were washed with PBS and treated with 5%
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sodium hypochlorite for 5 min. After washing the plate with PBS buffer and drying, the
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resorption pits were observed under a light microscope. Quantification of resorbed areas
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was performed using the ImageJ program.
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Retrovirus preparation and infection
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Retrovirus preparation and infection were conducted as described in ‘Materials and
46
Methods’. Briefly, to obtain retroviral particles, plasmids of pMX-IRES-GFP (the
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control) and pMX-c-Fos-GFP were transfected into Plat-E cells using Lipofectamine
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2000 reagent. After viral particles were collected from the culture medium for 48 h,
2
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BMMs were incubated with those in the presence of M-CSF (30 ng/ml) and polybrene
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(10 μg/mL) for 8 h. For the osteoclast formation assay, BMMs were treated with
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RANKL (10 ng/ml), M-CSF (30 ng/ml) and praeruptorin A (10 μM) for 4 days.
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Western blot analysis
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Cytoplasmic or nuclear protein fractions were prepared using a NucBuster Protein
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Extraction kit (Novagen, Germany). After protein quantification, Western blot analysis
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was performed as described in ‘Material and Methods’. Antibodies against p-IκBα,
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IκBα and NF-κB/p65 were purchased from Santa Cruz Biotechnology (CA). Antibody
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against Lamin B1 was obtained from AbFrontier (Seoul, Korea). Lamin B1 was used
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for the loading control of nuclear proteins.
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Statistical analysis
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All quantitative values are presented as mean ± SD. Statistical differences were
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analyzed using Student’s t-test. A value of p < 0.05 was considered significant.
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Supporting Information Legends
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Figure S1. Effect of praeruptorin A on cell spreading during RANKL-induced
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osteoclast differentiation. BMMs (1  104 cells/well) were seeded in a 96-well plate,
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treated with the vehicle (0.1% DMSO) or praeruptorin A (10 μM) for 2 h in the
77
presence of M-CSF (30 ng/ml), and incubated with RANKL (10 ng/ml) for 1 and 3 days.
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Then, cells were fixed, permeabilized, washed, and incubated with 10 μg/ml Hoechst
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33342.
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Figure S2. Effect of praeruptorin A on RANKL-induced osteoclast differentiation for
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the indicated periods. BMMs were cultured with praeruptorin A (10 M) for various
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times periods (indicated the black arrow) in the presence of M-CSF and RANKL. After
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TRAP staining, TRAP-positive multinuclear cells (MNCs; nuclear number > 3) were
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counted *, P < 0.05; **, P < 0.01; *** P < 0.001.
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Figure S3. Effect of praeruptorin A on the formation of actin rings during osteoclast
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differentiation. BMMs (1  104 cells/well) were seeded in a 96-well plate, treated with
89
the vehicle (0.1% DMSO) or praeruptorin A (10 μM) for 2 h in the presence of M-CSF
90
(30 ng/ml), and incubated with RANKL (10 ng/ml) for 4 days. Then, cells were fixed,
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permeabilized, washed, and stained with Hoechst 33342 and phalloidin-FITC for
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nucleus and actin rings, respectively.
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Figure S4. (A) Effect of age, strain or sex on anti-osteoclastogenic action of
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praeruptorin A. BMMs were isolated from mice (male and female ICR strain, 5-week
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old; male and female C57BL6/N strain, 5-week old and 8-month old) and cultured with
4
97
praeruptorin A in the presence of M-CSF and RANKL for 4 days. Osteoclast
98
differentiation was visualized by TRAP staining. (B) Effect of age, strain or sex on anti-
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osteoclastogenic action of praeruptorin A. BMMs were isolated from mice (male and
100
female ICR strain, 5-week old; male and female C57BL6/N strain, 5-week old and 8-
101
month old) and cultured with praeruptorin A in the presence of M-CSF and RANKL for
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4 days. After TRAP staining, TRAP-positive multinuclear cells (MNCs; nuclear number
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> 3) were counted. TRAP activity and cell viability were also evaluated. *, P < 0.05; **,
104
P < 0.01; *** P < 0.001.
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Figure S5. Anti-resorptive activity of praeruptorin A. (A) After co-culturing BMMs
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with osteoblasts for 7 days, multinucleated osteoclasts were replated on BioCoat
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Osteologic MultiTest slides and after 2 h incubation, cells were further incubated with
109
praeruptorin A and RANKL for 6 h. Then, cells were stained for TRAP (upper images).
110
(B) TRAP-positive multinucleated cells were counted. (C) After removing cells, the
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resorption pits (indicated by asterisks in bottom images) were observed under a light
112
microscope. The relative resorbing areas were evaluated using the ImageJ program. ***
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P < 0.001
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Figure S6. Effect of c-Fos on anti-osteoclastogenic action of praeruptorin A. BMMs
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were infected with retroviruses harboring the control GFP or c-Fos-GFP vectors.
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Transduced BMMs were cultured with RANKL (10 ng/ml) and M-CSF (30 ng/ml) in
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the presence of praeruptorin A (10 μM) or the vehicle (0.1% DMSO). (A) After
119
incubation for 2 days, GFP expression was visualized under a fluorescence microscope
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(left images). After 2 additional days, mature TRAP-positive multinucleated osteoclasts
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were visualized by TRAP staining (middle and right images). TRAP-positive cells
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(nuclear number > 3) were counted as osteoclasts (B), and TRAP activity was measured
123
at 405 nm (C). *, P < 0.05; **, P < 0.01; *** P < 0.001
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Figure S7. Effect of praeruptorin A on RANKL-induced activation of NF-κB signaling
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pathway. BMMs were treated with praeruptorin A (10 μM) for 30 min, stimulated with
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RANKL (10 ng/ml) for the indicated time. The expression levels of molecules in
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cytoplasmic or nuclear protein fractions were evaluated by Western blot analysis. Actin
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and lamin B1 were used for the loading control of cytosolic and nuclear proteins,
130
respectively. Densitometric analysis was performed using ImageJ software and the
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relative, normalized ratios of IB/actin, p-IB/actin, cytosolic p65/actin or nuclear
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p65/lamin B1 were presented.
6