Research Paper Volume 14, Issue 10 pp 4281—4304

Figure 8. CDK4/6 inhibitor Palbociclib induces senescence in MEF cells and mimicks AhR deficiency. Embryonic fibroblasts were cultured for 48 h, then plated at 4 x 10⁵ cells per 10-cm plate. Two days later, cells were treated with 4 μM CDK4/6 inhibitor Palbociclib/PD-0332991 for 8 days. (A) Bright-field microscopy of AhR+/+ and AhR−/− MEFs untreated or treated with Palbociclib. (B) Cell senescence measured as SA-β-Gal activity in AhR+/+ and AhR−/− MEFs. 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 Palbociclib treated AhR+/+ and AhR−/− MEFs. (D) AhR mRNA level was determined by RT-qPCR in AhR+/+ MEFs under both experimental conditions using oligonucleotides indicated in Supplementary Table 1. Determinations were done after 8 days of treatment with Palbociclib or vehicle (control). (E–G) mRNA levels 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).