Human AC16 Cardiac Cells under Doxorubicin Treatment

AC16 acquired from Millipore and cultured in DMEM/F12 with 10% FBS. For the mass spectrometry experiment, the AC16 cells were switched to Fibroblast Growth Medium (FGM-3) with supplements (Promocell) then treated with 0.1 µM doxorubicin for 24 hours, then co-cultured through non-contact transwells with primary human ventricular fibroblasts (Promocell) with or without TGFb stimulation for 24 hours. The induction of senescence with doxorubicin and lack of spontaneous senescence was verified using p21 immunoblots. For mass spectrometry, 25 µg of proteins were reduced, alkylated, and digested with trypsin using a filter-assisted protocol on a Pierce 10 K MWCO column. The peptides were then labeled using Tandem Mass Tag (TMT) 10-plex reagents (Thermo), combined, and separated by high-pH reversed phase separation (Thermo), then analyzed on an Orbitrap HF mass spectrometer.