Policosanol-induced osteoblast differentiation is mediated by INSIG. (a) Cells were treated with 50 μg/mL policosanol for 4 days. qPCR was performed using total RNA isolated from MC3T3-E1 cells. (b) Western blotting assay was performed using total protein isolated from 50 μg/mL policosanol-treated cells. (c,d) Cells were transfected with the INSIG1 or -2 overexpression vector, and 48 h after transfection expression of mRNA encoding INSIG1 and -2 were measured by qPCR. (e) Western blotting assay was performed using total protein isolated from INSIG1 and -2 overexpression vector-transfected cells. (f) Cells were co-transfected with the INSIG1 or -2 overexpression vector, and Runx2-luc vector. At 48 h after transfection, Runx2 luc-activity was measured by luciferase assay. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the untreated control. Data represent the mean ± SD of three individual experiments.