Protein Extraction and Immunodetection

Protein extraction and immunodetections were performed as previously described [62 (link)], using ECL Select detection reagent (GE Healthcare) and anti-p53 (DO-1) anti-RelA/p65 (C-20) anti- p21 (C19), anti-GAPDH (6C5) (Santa Cruz Biotechnology, Heidelberg, Germany). When appropriate, nuclear and cytoplasmic fractionation was performed. MCF7, A549 and H1299 cell lines were seeded on 100mm Petri dishes and treated at 80% confluence with Doxo, TNF⍺, BAY or the combination of the drugs for 16 hours. Cells were harvested and cytoplasmic and nuclear proteins were extracted using NE-PER Nuclear and Cytoplasmic Extraction Kit (Pierce, ThermoFisher Scientific), following the instructions provided by the manufacturer. 20 μg of nuclear and cytoplasmic extracts were loaded on a 12% poly-acrylamide gel and transferred to nitrocellulose membranes. Antibodies used for detection were: anti-Histone H3 (clone #: ab1791, AbCam, Milan, Italy) and anti-Lamin A/C (clone #: 2032, Cell Signaling, Milan, Italy) used as nuclear loading control, and anti-GAPDH used as cytoplasmic loading control.