Fig. 7
From: SIRT7 has a critical role in bone formation by regulating lysine acylation of SP7/Osterix
SIRT7 regulate OSX lysine propioylation. a Detection of acetylated or propionylated OSX. Calvarial osteoblasts differentiated for 6 days were treated with 10 mM NAM for 24 h or with 1 μM TSA for 6 h. After immunoprecipitation, acetylated and propionylated OSX were detected by WB. A long exposure time was required to detect acetylated OSX. Arrowhead and asterisk indicate OSX and non-specific protein, respectively. b,c Effect of Sirt7 deficiency on acetylation and propionylation of OSX. Protein lysates of osteoblasts differentiated from bone marrow mesenchymal stem cells (b) or of calvariae (c) were subjected to immunoprecipitation, after which acetylated and propionylated OSX were detected by WB. d Effect of SIRT7 overexpression on propionylation of OSX. Sirt7 KO MEF were transfected with the 3× HA-OSX expression plasmid and the SIRT7 or SIRT7H188Y expression plasmid, followed by treatment with 50 mM Na-Prop for 16 h. Propionylation of OSX was assessed by immunoprecipitation and WB. e, f Detection of propionylated OSX-mutants. MEF were transfected with the 3× HA-OSX or 3× HA-OSX (K41,45,46R) expression plasmid, after which cells were treated with 50 mM Na-Prop for 16 h (e). The 3× HA-OSX or 3× HA-OSX (K368R) expression plasmid combined with the SIRT7 or empty expression plasmid were transfected into Sirt7 KO MEF, which were subsequently treated with 50 mM Na-Prop for 16 h (f). Propionylation of OSX was assessed by immunoprecipitation and WB. g Impact of propionylation on transactivation activity of OSX. MC3T3-E1 cells were transfected with the GAL4DBD-OSX expression plasmid, and then were treated with 10, 30, 50 mM Na-Prop for 24 h. Subsequently, cells were transfected with the 5× GAL4-luciferase reporter and the reporter assay was performed after 24 h (n = 5 each). h Luciferase assay using recombinant OSX protein. OSX protein was transfected into MC3T3-E1 cells, followed by transfection of the Col1a1 enhancer/promoter-driven luciferase reporter after 2 h. The reporter assay was performed at 18 h after luciferase transfection (left) (n = 4 each). Transfected proteins were confirmed by WB (right). WB, western blotting; IP, immunoprecipitation; KAc acetyl lysine, KProp propionyl lysine. Data are shown as the mean ± SEM. Statistical significance was determined by Student’s t-test. *p < 0.05 vs. 0 mM Na-Prop (g)
