Research Paper Volume 8, Issue 12 pp 3450—3467

Figure 4. p53C124S mutant induces atrophy of C2C12 myoblasts. (A) C2C12 myoblasts were transfected with pcDNA3.1 vector containing cDNA for wild type p53 (Wt-p53), three single p53 mutants in DBD (p53C277S, p53C275S, p53C124S) or with empty vector (Mock). After 24 h from transfection C2C12 cells were differentiated for 4 days. Total RNA was isolated and relative mRNA levels of MyoD, PAX7 and Myogenin were analyzed by RT-qPCR. Data are expressed as means ± S.D. All the values were significantly different with respect to Mock day 0/4 (n=3, p<0.05; *p<0.05 was significantly decreased with respect Mock day 0). (B) Cells were counted by Trypan Blue exclusion. Data are expressed as means ± S.D. All the values were significantly different with respect to day 0 (n=4 *p<0.05). (C) Dead cells were counted by Trypan blue exclusion. Data are expressed as means ± SD (n=4, *p<0.001 vs Mock-, Wt-p53-, p53C277S- and p53C275S-day 0/4 cells). (D) Total RNA was isolated and relative mRNA levels of MuRF-1 and Atrogin-1 were analyzed by RT-qPCR. Data are expressed as means ± S.D. (n=3; *p<0.05 vs Mock-, Wt-p53-, p53C277S- and p53C275S-day 0/4 cells). (E) ChIP assay was carried out on cross-linked nuclei from Mock, Wt-p53, p53C277S, p53C275S and p53C124S cells at day 4 of myogenesis using p53 antibody followed by qPCR analysis of p53RE. Dashed line indicates the value of IgG control. Data are expressed as means ± S.D. (n=3; *p<0.001 vs Mock-, Wt-p53-, p53C277S- and p53C275S-day 4 cells).