Research Paper Volume 14, Issue 10 pp 4281—4304

Figure 6. Lack of AhR enhances in vitro cellular senescence in mouse embryonic fibroblasts. (A) Senescence profiles of AhR+/+ and AhR−/− MEFs at the indicated passages as determined by the level of SA-β-gal activity analyzed by FACS using the β-galactosidase fluorescent substrate C12FDG. Results are normalized to wildtype MEFs at passage 2. (B) Representative flow cytometric profiles of senescent cells stained with C12FDG in AhR+/+ and AhR−/− MEFs at passage 5. (C) SA-β-Gal activity in AhR+/+ and AhR−/− MEFs at passage 5 as determined by staining using X-Gal as substrate. (D) AhR mRNA expression was determined by RT-qPCR at the indicated cell culture passages. (E–G) mRNA expression of senescence driver genes p16^Ink4a (E), p21^Cip1 (F) and Trp53 (G) were determined in AhR+/+ and AhR−/− MEFs by RT-qPCR using the oligonucleotides indicated in Supplementary Table 1. Gapdh was used to normalize target gene expression (△Ct) and 2^−△△Ct to calculate changes in mRNA levels with respect to wild type or untreated conditions. Data are shown as mean + SD (^**P < 0.01; ^***P < 0.001).