Palbociclib hy a0065

IMR90 human embryo lung primary fibroblasts were obtained from ECACC. FF292 human adult primary dermal fibroblasts were a gift from Marisol Soengas, CNIO, Madrid. IMR90 ER:RAS fibroblasts (expressing the ER:RAS cassette) have been previously described (Adrados et al., 2016 (link)), FF292 ER:RAS fibroblasts were generated with the same strategy. Mouse embryo fibroblasts were obtained from E13.5 embryos. All the cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) with 10% fetal bovine serum (FBS; Gibco), containing penicillin and streptomycin, and were used at low passage. To induce senescence, cells were treated with palbociclib (HY‐A0065; MedChemExpress) at 10 μM for 9 days or bleomycin (bleomycin sulfate; Mylan) at 10 µM for 24 h and analyzed 6 days later. Fibroblasts were also irradiated with 10 Gy and analyzed 10 days later. Doxycycline (D1822; Sigma) was used at 1 µM. TNF‐α (300‐01A; PeproTech) was used at 10 and 20 ng/ml for 48 h. Human BMP2 protein (355‐BM‐010/CF; R&D Systems) was used at 50 μg/ml for 24 h. Human BMP7 protein (354‐BP‐010/CF; R&D Systems) was used at 400 μg/ml for 24 h. For TGF‐β treatments, cells were grown in DMEM with 0.01% FBS for 48 h prior to the addition of TGF‐β1 (100‐21; PeproTech).

T47D and MCF-7 cell lines were obtained from American Type Culture Collection (ATCC) and were maintained in DMEM (Cell Technologies) with 10% foetal bovine serum at 37 °C in a humidified atmosphere containing 5% CO₂. These two cell lines have been validated by the short tandem repeat (STR) method. Inhibitors used in the treatment of cells, including palbociclib (HYA0065) and rapamycin (HY-10219), were purchased from MedChemExpress (MCE).