Fig. 7.

Has2 expression in VSMCs and femoral artery. a Starved WT, SMIRKO, or SMIGF1RKO VSMCs were stimulated with 10 nM insulin, and the gene expression of Has2 was determined by qPCR (0 h, n = 4 for each group; 0.5 h, n = 3 for each group; 1 h, n = 3 for each group; 2 h, n = 4 for each group; 4 h and 8 h, n = 4 for WT and SMIGF1RKO, n = 3 for SMIRKO; 24 h, n = 3 for WT and SMIGF1RKO; n = 2 for SMIRKO). b Has2 protein expression in VSMCs treated with insulin. c Hyaluronan in cultured medium. The hyaluronan content before and 12 h after 10 nM insulin stimulation was determined by ELISA. d The femoral arteries were harvested at 9 days after wire injury, and the expression of Has2 gene was determined by qPCR (IR^flox/flox sham, n = 6; IR^flox/flox injury n = 7; SMIRKO sham, n = 4; SMIRKO injury, n = 8; IGF1R^flox/flox sham, n = 6; IGF1R^flox/flox injury, n = 9; SMIGF1RKO sham, n = 3; SMIGF1RKO injury, n = 5). e, f Hyaluronan staining in wire-injured femoral artery. Hyaluronan staining was performed on feoral arteries at 4 weeks after wire injury (arrow indicates neointima. IR^flox/flox, n = 8; SMIRKO, n = 8; IGF1R^flox/flox, n = 9; SMIGF1RKO, n = 7). g VSMCs were infected with AdFoxO1 CA or AdGFP and then stimulated with insulin for 4 h. The expression of Has2 gene was determined by qPCR (n = 3 per group). h VSMCs were treated with rapamycin (10 ng/ml) for 1 h and then stimulated with insulin for 4 h. Expression of Has2 gene was determined by qPCR (n = 3 per group). i The expression of Has2 in VSMCs was knockdown by siRNA, and cellular proliferation was determined by EdU incorporation (n = 8 for each group). j Starved VSMCs were pretreated with 1 mM 4-MU for 4 h and then stimulated with 100 nM insulin for 24 h. The cell proliferation was determined by measuring EdU-positive cells by flow cytometry (n = 3 per group). The data are mean ± SEM. Two-tailed t test or two-way ANOVA with a post hoc test. Source data are provided as a Source Data file