Fig. 5

SIRT7 interacts with OSX and promotes its transcriptional activity. a Transcriptional activity of OSX in Sirt7 KO and WT osteoblasts. Cells were transfected with the GAL4DBD-OSX expression plasmid and the 5× GAL4-luciferase reporter plasmid, and luciferase activity was determined 24 h after transfection. b Transcription of the Osx (left) and Col1a1 (right) enhancer/promoter-driven luciferase reporters in MC3T3-E1 cells. After the indicated siRNA was introduced for 72 h, cells were transfected with the OSX expression plasmid and the indicated luciferase reporter plasmid. The reporter assay was performed 24 h later. n = 3 each. c, d Co-immunoprecipitation assay detecting the interaction between HA-SIRT7 and OSX in HEK293T cells (c), and that between endogenous SIRT7 and OSX in primary calvarial osteoblasts differentiated for 6 days (d). e Mapping the site of interaction between SIRT7 and OSX. Pull-down assay of Halo-SIRT7-FLAG deletion mutants was performed with lysate of OSX-HA overexpressing HEK293T cells. Expression of Halo-FLAG and Halo-SIRT7-FLAG proteins (M1–4 deletion mutants; amino acids 1–90 (M1), 91–210 (M2), 211–332 (M3), and 333–402 (M4)) was determined by WB. f Effect of SIRT7 overexpression on transcriptional activity of OSX. MC3T3-E1 cells were transfected with the indicated siRNA, and 72 h later were transfected with the GAL4DBD-OSX expression plasmid, the 5× GAL4-luciferase reporter plasmid, and SIRT7 or SIRT7^H188Y expression plasmid. The reporter assay was performed after 24 h. n = 3 each. g GAL4DBD-OSX protein level analyzed by WB under the conditions in h. WB western blotting; IP, immunoprecipitation. Data are shown as the mean ± SEM. Statistical significance was determined by Student’s t-test. *p < 0.05