Research Paper Volume 14, Issue 10 pp 4281—4304

Figure 7. Senolytic agent Navitoclax restores wild-type mRNA levels of senescence driver genes in AhR−/− MEFs. Embryonic fibroblasts at P4 or P5 were treated with vehicle or 10 μM Navitoclax for 48 h. (A) Bright-field microscopy of AhR+/+ and AhR−/− MEFs untreated or treated with Navitoclax. (B) Cell senescence measured as percentage of SA-β-Gal activity in MEF cells of both genotypes. SA-β-Gal activity was analyzed by FACS using the β-galactosidase fluorescent substrate C12FDG. Results are normalized to vehicle-treated wild-type MEFs. (C) X-Gal staining in untreated and Navitoclax-treated AhR+/+ and AhR−/− MEFs. (D) AhR mRNA expression was determined by RT-qPCR in both experimental conditions using the oligonucleotides indicated in Supplementary Table 1. (E–G) mRNA expression of senescence driver genes p16Ink4a (E), p21Cip1 (F) and Trp53 (G) was determined in AhR+/+ and AhR−/− MEFs by RT-qPCR using 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 < 0.05; ^**P < 0.01; ^***P < 0.001).