Fig. 1

Aim2 knockout affects the morphology, replicative potential and senescence of murine VSMC. VSMC were isolated from WT and Aim2^−/− mice and pooled. Each pool contained VSMC of 3 animals. a VSMC derived from Aim2−/− mice are larger, rounded and contain big vacuoles. The picture shows representative cultures of passage 3 from n = 6 (Aim2−/−) and n = 7 (WT) VSMC pools. b VSMC derived from Aim2−/− mice (n = 9 pools) undergo faster replicative senescence than WT VSMC (n = 3 pools). c Viability of VSMC was determined by WST1-proliferation assay after proliferation of the cells for 24, 48, 72 and 96 h in normal growth medium. d-e Analysis of mRNA expression of the senescence marker Cdkn2a/p16 (d) and Acta2 (e) was determined by real-time RT-PCR from VSMC grown in normal growth medium or in mineralization medium (OM). Data show the mean and SEM derived from 3 pools of proliferating (passage 3–4) and senescent (passage 7–12) VSMC. Senescence was defined as no proliferation for > 2 weeks. Data were tested by one-way ANOVA. ns: not significant, *: P < 0.05; ** P < 0.01; ****: P < 0.0001. f Western blot showing expression of p16ink4A and α-SMA in VSMC pools (derived from n = 2–3 animals per pool) of Aim2−/− versus WT mice. Numbers represent the relative protein level normalized to GAPDH. Expression level in the spleen of WT mice was set as 1.0