Mammalian cells undergo ploidy changes and down-regulation of SAC and cohesin components as they approach SEN in culture.
(a) Two-chromosomes FISH analysis of ploidy of human primary fibroblasts (HPF) was performed at early (PD42), mid (PD56) and late PDs/SEN (PD74). Plots depict the percentage of cells that were found diploid (2n, black) or not-diploid (Not 2n, gray). (b) Two-chromosomes FISH analysis of ploidy performed on mouse embryonic fibroblasts (MEFs) at early (PD04) and late PDs/SEN (PD10). (c) Gene expression analysis depicting mRNA levels of BUB1B, BUB1, SMC1A and BUB3 in non-mitotic quiescent cells (QUI-white) and SEN cells (SEN-grey) relative to proliferating cells (PRO-black). Down-regulation of the tested genes is statistically significant only in SEN cells. (d,e) Lentiviral delivery of shRNAs results in knockdown of BUB1 and SMC1A in IMR-90 fibroblasts as detected by western blotting. (d) Knockdown of BUB1 and (e) SMC1A at the protein level (EV = Empty vector infected cells). TUB4A was used as loading control. (*) Indicates significant differences (p < 0.05) from PRO cells tested by One-way ANOVA. Data are expressed as mean ± SD (n = 3).