Foxa1

Paraffin-embedded slides were deparaffinized and rehydrated followed by antigen retrieval using citric acid buffer. Endogenous peroxidase was deactivated by H₂O₂. Slides were blocked using 10% goat serum and incubated with the corresponding primary antibodies overnight at 4 °C. After incubation with secondary antibodies for 45 min at room temperature, DAB staining (Thermo Fisher Scientific) was used to detect the antigen–antibody binding. The primary antibodies used were: GABPA (Cell Signaling Technology) and FoxA1 (Santa Cruz Biotechnology). The slides were examined by two of the coauthors (YG and DX) and mean values of GABPA and FoxA1 positive cells were presented based on the results from two observers. For each slide, a total of 200 cells in two fields were analyzed.

Whole cell extracts were prepared in 50 mM Tris-HCl, 150 mM NaCl, 1% NP40, 10 mM NaF, 10% Glycerol, supplemented with protease inhibitor cocktail (Roche, Cat. No. 04693116001) and quantitated using a BCA approach. 60 μg of protein, or 40 μl of conditioned media, was run on 10% acrylamide gels and transferred onto nitrocellulose membrane. Western blots were imaged on radiography film or by a LI-COR Odyssey system. actin or vinculin served as loading controls. Primary antibodies: phospho-Akt (Ser473; Cell Signaling Technology, Cat. No. 4060S), pan-Akt, (Life Technologies, Cat. No. 44609G), androgen receptor (Santa Cruz Biotechnology, Cat. No. sc-816), SEMA3C (Santa Cruz Biotechnology, Cat. No. sc-27796), GATA2 (Santa Cruz Biotechnology, Cat. No. sc-9008), FOXA1 (Santa Cruz Biotechnology, Cat. No. sc-6553), POU2F1 (Santa Cruz Biotechnology, Cat. No.s sc-232, sc-8024; Cell Signaling Technology, Cat. No. 4428S), actin (Sigma-Aldrich, Cat. No. A2066), and vinculin (Sigma-Aldrich, Cat. No. V4505). Secondary antibodies: anti-rabbit alexa fluor 680 (Invitrogen, Cat. No. A21109), anti-mouse alexa fluor 680 (Invitrogen, Cat. No. A21058), anti-goat alexa fluor 680 (Invitrogen, Cat. No. A21084), anti-rabbit HRP (Dako, Cat. No. P0448), anti-mouse HRP (Dako, Cat. No. P0447), and anti-goat HRP (Dako, Cat. No. P0160).

The following primary antibodies were used at the stated titrations for immunoblotting (IB), immunofluorescence (IF) and immunohistochemistry (IHC): β-actin (ACTB; Sigma-Aldrich, AC-15) (IB – 1:250,000); claudin 3 (Life Technologies, Z23.JM) (IB – 1:4000); claudin 4 (Life Technologies, 3E2C1) (IB – 1:1000); claudin 5 (Life Technologies, 4C3C2) (IB – 1:1000); claudin 7 (Life Technologies, ZMD.241) (IB – 1:1000); cytokeratin 5 (CK5) (Abcam, SP27) (IHC – 1:100); cytokeratin 5 (CK5) (The Binding Site, PH607) (IF – 1:100); cytokeratin 7 (CK7) (Novocastra, OV-TL12/30) (IHC – 1:400, IF – 1:40); cytokeratin 13 (CK13) (Abnova, 1C7) (IHC – 1:500, IF – 1:500); cytokeratin 14 (CK14) (Serotec, LL002) (IHC – 1:1200); cytokeratin 14 (CK14) (ICRF, LL001) (IF – 1:5); cytokeratin 20 (CK20) (Novocastra, Kr20.8) (IHC – 1:200); Cytokeratin 20 (CK20) (Cymbus Bioscience, IT-Ks20.3) (IF – 1:100); ELF3 (Abcam, EPESER1) (IF – 1:1000, IB – 1:20,000); FOXA1 (Santa Cruz Biotechnology, Q-6) (IF – 1:200, IB – 1:500); FOXA1/2 (Santa Cruz Biotechnology, C-20) (IF – 1:200); GATA3 (Cell Signaling, D13C9) (IF – 1:800, IB – 1:1000); PPARγ (Cell Signaling, D69) (IF – 1:100, IB – 1:500).