Fig. 2

Aim2 knockout increases calcification of VSMC grown in mineralization medium. a Representative images of Alizarin Red S (ARS) stained VSMC. Proliferating and senescent cells were grown in mineralization medium (+ OM) or regular SMC growth medium (− OM). Cells were stained with ARS for visualization in triplicates at day 21. Bar: 40 μm. b Quantitative measurement of ARS staining. ARS was eluted from the cells and analyzed photometrically at 405 nm. Bars show the mean and SD from 3 measurements of VSMC pools. c-e Analysis of mRNA expression of Bmp4 (c), Tnfsf11/Rankl (d), and Runx2 (e), by real-time RT-PCR. Total RNA was extracted from VSMC grown in vitro with normal growth medium (−OM; n = 3 pools) or osteoblast mineralization medium (+ OM; n = 3 pools) at day 14. A.u. arbitrary unit; relative expression of target gene was normalized against expression of housekeeping genes (Actb, Gapdh and B2m). Bars represent the mean and SEM of three measurements from three pools each. a-e Data were tested by one-way ANOVA. ns: not significant, *: P < 0.05, **: P < 0.01, ****: P < 0.0001. f Concentration of murine sRANKL secreted into the supernatant, as determined by ELISA. Data show the mean and SEM of n = 3 independent experiments with different VSMC pools for each genotype