ABSTRACT: Transformed AC16 cells were acquired from Millipore and used at P10-15 and expanded in DMEM/F-12 with 10% FBS (Gibco). Primary human ventricular cardiac fibroblasts were acquired from Promocell and expanded in FGM-3 (Promocell) and used at P3-5. For the experiment, cells were separately placed on trans-wells/inserts (0.4 µm pore size, Greiner # 657641) at a density of 3,500 – 3,560 cells/cm2 for AC16 cells and plated in normal six-well tissue-culture coated plates at a density of 3,350 – 3,550 cells/cm2 for human ventricular cardiac fibroblasts. The cells are separately cultured in fibroblast growth medium FGM-3 (Promocell), which was tested in previous experiments to support the growth of AC16 cells for at least 48 hours. Once the AC16 cells achieved 40% confluency a 0.1 µM doxorubicin and vehicle FGM-3 media were prepared to replace the media from four six-well plates with transwell inserts. Once the primary human fibroblasts achieved >10% confluence, 5ng/mL of TGF-β1 and vehicle FGM-3 media were prepared and used to replace the media from six separate 6-well-plates. All cells were cultured at 37°C in 5% CO2. To initiate co-culture, the media in the AC16 cells were removed then the cells were washed with PBS twice; 1.5mL of FGM-3 was added to each trans-well. The media for the six fibroblast plates were changed with fresh corresponding TGF-β1 or vehicle treated FGM-3 media. The transwells were nested into each well of the fibroblast plates following the experimental design. Primary cardiac ventricular fibroblasts were lysed using RIPA buffer and Protease Inhibitor (Pierce Halt) followed by sonication (Bioruptor) to solubilize proteins. Protein concentration in each sample was measured using BCA assay (Thermo) against a standard curve of BSA. For mass spectrometry, proteins were digested using a filter-assisted sample preparation protoco: 25 µg protein from each sample was added to 10 kDa MWCO 0.5 mL volume PES protein concentrator (Thermo Scientific), washed with 250 µL of 8 M urea centrifuged (10 min, 22°C, 14,000 g) 2x to remove detergent, then exchanged to 300 µL 100 mM TEAB 2x followed by centrifuged (10 min, 22°C, 14,000 g), reduced with 10 mM TCEP and alkylated with 40 mM CAA, digested with sequencing grade trypsin (Promega) O/N at 37 °C. Peptides were labeled with TMT 10-plex tags for 1 hr, followed by quenching with 1 µL 5% hydroxylamine. The samples were dried, desalted with C18 clean-up columns (Thermo), then fractionated using the Pierce High pH Reversed-Phase Peptide Fractionation Kit (Thermo) following manufacturer’s protocol. The samples were analyzed on a Q-Exactive HF Orbitrap mass spectrometer coupled to an Easy-nLC 1200 liquid chromatograph (Thermo) in data-dependent acquisition mode using conventional settings. Raw spectra were converted to mzML format using ThermoRawFileParser, then searched using Comet v.2022_01 against the UniProt SwissProt human protein sequence canonical + isoform database appended with common contaminant protein sequences (retrieved 2023-03-22 using Philosopher; 42,435 forward entries). Post-processing and calculation of false discovery rate of identification was performed using Percolator (crux v.4.1 distribution) [8], with 1% FDR accepted for ID. Spectrum TMT intensity was extracted using pyTMT v.0.4.1.
