SRT2183 inhibits RANKL-induced osteoclastogenesis and pit formation in s<i>irt1</i>^<i>-/-</i> BMMs.

<p>(A) Sirt1 expression in WT- and in <i>Sirt1</i>^<i>-/-</i>-derived osteoclasts. PCR amplification of exons 1–9 of the s<i>irt1</i> gene (left panel) and Western blot analysis with Sirt1 antibody (right panel) demonstrates complete loss of Sirt1 protein in osteoclasts obtained from <i>Sirt1</i>^<i>Δ/Δ</i> (<i>Sirt1</i>^<i>-/-</i>) mice. (B) The effect of SRT2183 on osteoclast differentiation in <i>Sirt1</i>^<i>-/-</i>-derived BMMs. BMMs were inducted to osteoclastogenesis with RANKL in the presence or absence of SRT2183. TRAP staining performed 4 days post induction. (C) The effect of SRT2183 on pit formation in <i>Sirt1</i>^<i>-/-</i>-derived BMMs stimulated with RANKL. An eroded area (left panel) and pit formation assay (right) are shown. (D) The effect of SRT2183 on p65 acetylation (Lys310). Western blot analysis of p65K310 ac and p65 in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation. (E) The effect of SRT2183 on AMPKα phosphorylation (Thr172). Western blot analysis of pAMPKα and AMPKα in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation. (F) The effect of SRT2183 on IκBα protein level. Western blot analysis of IκBα and GAPDH in SRT2183- and vehicle-treated BMMs 24 hours post RANKL stimulation. (G-H) The effect of SRT2183 on Sirt3 protein (G) and gene expression (H). Western blot analysis of Sirt3 and GAPDH in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation (G). Gene expression analysis by quantitative Real-Time PCR 4 days post RANKL stimulation is shown. Results are relative to <i>Polr2a</i> (H). (I) The effect of SRT2183 on superoxide dismutase 2 (Sod2) Lys68 acetylation. Western blot analysis of acetylated (ac) Sod2K68 and Sod2 in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation. Data are Mean ± SEM (n = 3 independent experiments), analyzed by paired Student's <i>t</i>-test paired (C) or one-sample Student's <i>t</i>-test (H-I); ***<i>P</i><0.001, ****<i>P</i><0.0001, versus vehicle-treated BMMs. Magnification X40; scale bar 1mm.</p

SRT2183 inhibits RANKL-induced NFATc1 activation in bone marrow-derived macrophages (BMMs).

<p>(A) The effect of SRT2183 on NFATc1 protein level. Western blot analysis of NFATc1 and HSP90 in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation. NFATc1- nuclear factor of activated T-cell cytoplasmic 1. (B) The effect of SRT2183 on DC-STAMP protein level. Western blot analysis of DC-STAMP and GAPDH in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation. DC-STAMP- dendritic cell-specific transmembrane protein. (C) The effect of SRT2183 on mRNA expression of osteoclast markers and fusion-related genes. SRT2183 or vehicle were co-administrated with RANKL. Gene expression analysis by quantitative Real-Time PCR 4 days post RANKL stimulation is shown. Results are relative to <i>GAPDH</i>. Data are Mean ±SEM (n = 3 independent experiments), analyzed by one-sample Student's <i>t</i>-test, *<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001 compared to vehicle-treated BMMs.</p

SRT2183 inhibits RANKL-induced osteoclastogenesis in bone marrow-derived macrophages (BMMs).

<p>(A) Chemical structure of SRT2183. (B-C) TRAP staining of SRT2183 or vehicle-treated BMMs inducted to osteoclastogenesis 4 days post RANKL stimulation (B). Total number of osteoclasts (left panel) and number of multinuclear cells (right) are shown (C). (D) The effect of SRT2183 on pit formation by RANKL-induced BMMs. A pit formation assay (left panel) and eroded area (right) are shown. (E) Time course of the effect of SRT2183 or vehicle (DMSO) administration on osteoclast differentiation. SRT2183 or a vehicle was added in the proliferation (a), differentiation (b, c) or maturation (c, d) phase. Arrows indicate periods of treatments with SRT2183. TRAP staining is shown. (F) The effect of SRT2183 on cell proliferation during the proliferation phase. SRT2183 or a vehicle were co-administrated with M-CSF for 72 hours on day of plating. (G-H) The effect of SRT2183 on cell viability and apoptosis during the proliferation phase (G) and the differentiation and maturation phase (H). SRT2183 or vehicle were co-administrated with M-CSF for 72 hours on day of plating (G) or with RANKL 3 days post plating (H). The graphs illustrate fold change in Caspase 3 activity and the percent change in living cells with time. Data are Mean ±SEM (n = 3 independent experiments), analyzed by 2 way ANOVA with nuclei number and treatment as the independent variables followed by Sidak's post-hoc correction (C), paired Student's <i>t</i>-test (D), one-sample Student's <i>t</i>-test (F-H), *<i>P</i><0.05; **<i>P</i><0.01 compared to vehicle-treated BMMs. Magnification X40; Scale bar 1mm.</p

SRT2183 activates AMPK and deacetylates RelA/p65 K310 in RANKL-induced BMMs.

<p>(A) The effect of SRT2183 on AMPKα phosphorylation (Thr172). Western blot analysis of pAMPKα and AMPKα in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation. p- phosphorylated; AMPKα- AMP-activated protein kinase alpha. (B) The effect of SRT2183 on ACC phosphorylation. Western blot analysis of pACC and ACC in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation. p- phosphorylated; ACC-Acetyl CoA Carboxylase. (C) The effect of SRT2183 on Sirt1 protein level in RANKL-stimulated osteoclasts. Western blot analysis of Sirt1 and HSP90 in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation. (D) The effect of SRT2183 on IκBα protein level. Western blot analysis of IκBα and GAPDH in SRT2183- and vehicle-treated BMMs 24 hours post RANKL stimulation. (E) The effect of SRT2183 on p65 acetylation (Lys310). Western blot analysis of p65K310 ac and p65 in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation. Data are Mean ±SEM (n = 3 independent experiments), analyzed by one-sample Student's <i>t</i>-test; *<i>P</i><0.05 versus vehicle-treated BMMs.</p