Anti p53

Anti-LEFTY, anti-Smad2, and anti-phospho-Smad2 at serine 255 (pSmad2) antibodies were purchased from Abcam (Cambridge, MA, USA). Anti- XIAP, anti-bax, anti-β-catenin, anti-HNF-1β, and anti-p27^Kip1 antibodies were bought from BD Biosciences (San Jose, CA, USA). Anti-p21^waf1, anti-cyclin D1, anti-p53, anti-bcl2, and anti-Ki-67 antibodies were purchased from Dako (Copenhagen, Denmark). Anti-cyclin A, and anti-cleaved caspase 3 antibodies were from Novocastra (Newcastle, UK), and Cell Signaling Technology (Danvers, MA, USA), respectively. CDDP and the anti-β-actin antibody were purchased from Sigma-Aldrich Chemicals (St. Louis, MO, USA). Recombinant transforming growth factor (TGF)-β1 was purchased from R&D Systems (Minneapolis, MN, USA).

Western blot analysis was carried out as described (Corno et al., 2017 (link)). Briefly, samples were fractionated by SDS-PAGE and blotted on nitrocellulose membranes. Blots were pre-blocked in PBS containing 5% (w/v) dried no fat milk, and then incubated overnight at 4°C with the following antibodies: anti-phospho-Akt (Ser473), anti-Akt (BD Science, Franklin Lakes, NJ, United States), anti-phospho-ERK1/2 (Thr202/Tyr204, Thr185/Tyr187), anti-ERK1/2, anti-AR (Millipore, Burlington, MA, United States); anti-Hsp90 (ac-Lys294) (Novus, Centennial, Colorado, United States), anti-Hsp90 (Santa Cruz Biotechnology, Dallas, TX, United States), anti-acetylated alfa-tubulin (Sigma-Aldrich, Milan, Italy), anti-Bax and anti-FLIP_L (Sigma-Aldrich, Milan, Italy), anti-p53 (Dako, Santa Clara, CA, United States), anti-cleaved caspase-3 (Asp175) and anti-cleaved caspase-7 (Asp198) (Cell Signaling, Danvers, MA, United States). Anti-vinculin (Sigma-Aldrich, Milan, Italy), anti-β-tubulin (Abcam, Cambridge, United Kingdom) or anti-actin (Sigma) antibodies were used as control for loading. Antibody binding to blots was detected by chemo-luminescence (Amersham Biosciences, Cologno Monzese, Italy). Three independent experiments were performed.

Immunohistochemistry (IHC) for P53, PAX8, FOXJ1, estrogen receptor and β-catenin was performed on 4-μm TMA sections using a BOND-MAX automated immunostainer and a Bond Polymer Refine Detection kit (Leica Microsystems, Wetzlar, Germany) according to the manufacturer’s guidelines. The primary antibodies were anti-P53 (DAKO, 1:1000), anti-PAX8 (Proteintech, 1:300), anti-FOXJ1 (Invitrogen, 1:100), anti-estrogen receptor (DAKO, 1:100), and anti-β-catenin (BD Transduction, 1:800). Estrogen receptor and β-catenin were considered positive when more than 10% of tumor cell nuclei were strongly stained.