Extended Data Fig. 5: SIRT2-KO^hep prevents against OVX-induced bone loss through upregulating LRG1 expression in hepatocytes and sEVs.

a, Heatmap of differentially expressed genes identified by RNA-seq using liver tissues from aged LoxP (n = 3) and SIRT2-KO^hep mice (n = 3). b, Western blot analysis of LRG1 protein expression in the livers and plasma sEVs of aged LoxP and SIRT2-KO^hep mice. n = 3 mice.one technical replicate of 3 biological replicates for each group. c, d, Western blot density analyzed by ImageJ and quantification analysis was shown. n = 3 mice, one technical replicate of 3 biological replicates for each group. e, Western blot analysis of LRG1 protein expression in the cytoplasm and supernatant-derived sEVs of NC and shSIRT2-AML12 hepatocytes. f, Western blot analysis of respective LRG1, Saa1/2 protein level in plasma, plasma sEVs and plasma removed sEVs (-sEVs). g, Western blot analysis of H4K16ac protein levels in the liver of aged LoxP and SIRT2-KO^hep mice. n = 3 mice.one technical replicate of 3 biological replicates for each group. h,ChIP analysis showing the enrichment of H4K16ac at the LRG1 proximal promoter region in NC and shSIRT2-AML12 hepatocytes using the primers p4 and p5, which are located at the distant upstream or downstream sites of LRG1 promoter and used as negative control. n = 3 biologically independent experiments. i, Uterine weight in each group after OVX or sham operation (Sham-LoxP-Ctrl mice: n = 7; Sham-SIRT2-KO^hep-Ctrl mice: n = 7; OVX-LoxP-Ctrl mice: n = 8; OVX-SIRT2-KO^hep-Ctrl mice: n = 8; and OVX-SIRT2-KO^hep-shLRG1 mice: n = 8). (one technical replicate of 7 (Sham-LoxP-Ctrl mice); 7 (Sham-SIRT2-KO^hep-Ctrl mice); 8 (OVX-LoxP-Ctrl mice); 8 (OVX-SIRT2-KO^hep-Ctrl mice) and 8 (OVX-SIRT2-KO^hep-shLRG1 mice) biological replicates for each group). j, The real-time observation of AAV8 liver-specific expression in each group was performed by BLI at 14th day after viral injection. k, Western blot analysis of LRG1 protein levels in the liver and plasma sEVs of each group. one technical replicate of 2 biological replicates for each group. Data are presented as mean ± SD. with biologically individual data points shown. P values are determined by unpaired two-tailed Student’s t-test (c, d, i), two-way ANOVA followed by Tukey’s test (h). n.s., not significant.

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