Proteome profiler array human apoptosis array kit

The Proteome Profiler™ Array Human Apoptosis Array Kit (R&D Systems) was used according to the manufacturer’s instructions 48 h after GapmeR treatment. Shortly, arrays were blocked for 1 h with array buffer and then incubated with cell lysates overnight at 4 °C. Arrays were incubated with Detection Antibody Cocktail for 1 h and subsequently with Streptavidin-HRP for 30 min at RT. Chemi Reagent Mix was used for visualization with the Amersham Imager 600 (GE Health care). Band intensities were quantified in ImageQuant TL (GE Health care).

The proteome profiler array—human apoptosis array kit (R&D Systems, Inc.) was used. UM‐UC‐3 and J82 were treated for 24 h and for 72 h with IC₅₀ dosage of quisinostat; DMSO treatment was used as control. Cells were detached and counted for a cell suspension concentration of 1 × 10⁷ cells/mL in lysis buffer. Protein concentration was assessed with the Pierce BCA protein assay (ThermoFisher Scientific) and 350 μg of proteins were used for the assay. Arrays and samples were subsequently processed according to the manufacturer's recommendation. For quantification, the intensity of the signal for each dot was assessed with the Image Lab Software (BioRad).

Total protein from HT29 and SW620 human colon cancer cell lines were extracted using 600 µL of radio immunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl, 150 mMNaCl, 1.0% TritonX-100, 0.5% sodium deoxycholate and 0.1% SDS) and supplemented with 10 mg of protease inhibitor cocktail (Roche, Basel, Switzerland). The extracted protein was then quantified using Bradford assay (Sigma, St. Louis, MO, USA). The proteome profiling assay was conducted to confirm the induction of cell death via apoptosis and the expressed protein levels were determined using the Proteome Profiler™ Array Human Apoptosis Array Kit (R&D Systems, Inc., Minneapolis, MN, USA), conducted according to the manufacturer’s protocol provided in the kit. The membrane was scanned using the ChemiDoc XRS (BioRad, Hercules, CA, USA) and analyzed using Image Lab™ Software version 6.0.0 (Bio-Rad Laboratories, Inc., Hercules, CA, USA).