E cadherin

β-actin (Sigma Aldrich mouse monoclonal anti-β-actin, A2228, 1:2000), 42 kDa. N-cadherin (BD Biosciences mouse monoclonal anti-N-cadherin, 610921, 1:2000) 120 kDa. E-cadherin (BD Biosciences mouse monoclonal anti-E-cadherin, 610181, 1:2000) 120 kDa. Vimentin (Sigma-Aldrich goat polyclonal anti-Vimentin, V4630, 1:1000) 58 kDa. S100A10(BD Biosciences mouse monoclonal anti-S100A10, 610070, 1:2000) 11 kDa. Annexin A2 (BD Biosciences mouse monoclonal anti-Annexin II, 610069, 1:2000) 36 kDa. GAPDH (Biochain mouse monoclonal anti-GAPDH, Y3322, 1:2000) 36 kDa. p-S6K (Cell signaling rabbit monoclonal anti-pS6K, 9205 S, 1:1000) 70 kDa. FOXC2 (Bethyl laboratories rabbit polyclonal anti-FOXC2, A302-383A, 1:1000) 65–70 kDa. PAI-1 (Cell signaling rabbit monoclonal anti-PAI-1 D9C4, 11907, 1:2000) 48 kDa. uPAR (Santa Cruz rabbit polyclonal anti-uPA H149, sc-10815, 1:300) 55 kDa. p-ERK (Erk1/2) (Cell signaling rabbit polyclonal anti-Erk1/2 (Thr202/Tyr204), 9101, 1:1000) 42, 44 kDa.

Eleven paired formalin-fixed tissue samples from our previous study (ALK1, ALK4, ALK5, ALK6, ALK14, ALK16, ALK17, ALK20, ALK22, ALK25, and ALK26) were used for IHC staining of two EMT biomarkers: E-cadherin (using mouse monoclonal E-cadherin 1:100, clone 36/E-cadherin; BD Biosciences, Breda, The Netherlands) and Vimentin (using mouse monoclonal Vimentin 1:100, clone sc-6260; Santa Cruz Biotechnology; Bioconnect, Huissen, The Netherlands).

Cell lysis and immunoblotting were essentially performed as described previously [8 (link),9 (link),10 (link)] with minor modifications. Proteins were fractionated by polyacrylamide gel electrophoresis on TGX Stain-Free FastCast gels (BioRad, Munich, Germany). Following blotting and antibody treatment, chemoluminescent detection of proteins was carried with a ChemiDoc XRS+ System with Image Lab Software (BioRad) with Amersham ECL Prime Detection Reagent (GE Healthcare, Munich, Germany). The signals for the proteins of interest were normalized to either the total amount of protein in the same lane (when using the TGX Stain-Free FastCast gels), or to bands for the housekeeping gene GAPDH. Significant differences (p < 0.05)-calculated with the unpaired two-tailed Student’s t test–denoted by bars above the respective cell lines. The antibodies used were Rac1b (#09-271, Merck Millipore, Darmstadt, Germany), E-cadherin (#610181) and Rac1 (#610650) (BD Transduction Laboratories, Heidelberg, Germany), GAPDH (14C10, #2118, Cell Signaling Technology, Frankfurt am Main, Germany), Vimentin (clone V9, #V6630, Sigma-Aldrich, Steinheim, Germany), and HA (clone 12CA5, Roche Diagnostics, Mannheim, Germany).

Immunoblot and immunofluorescence analysis were performed as previously described18 (link). Primary antibodies used were: SOX9 (AB5535, Millipore), phospho-SOX9 (ab59252, abcam), BMI1 (05-637, Millipore), E-cadherin (BD610181, BD Transduction Laboratories), Vimentin (M7020, DAKO), N-Cadherin (BD610920, BD Transduction Laboratories), phospho-S6 Ribosomal protein (Cell Signaling Technology^®, #4858) and β-actin (AC-15, Sigma). For Western blot detection of primary antibodies, we used HRP-linked antibodies (Santa Cruz Biotechnology) and detection was performed by chemiluminescence using NOVEX ECL Chemi Substrate (ThermoFisher). For immunofluorescence, secondary antibodies conjugated with fluorochromes were used and nuclear DNA was stained with Hoechst 33342 (Sigma). Images were obtained at a 40x magnification.

The antibodies and inhibitors used are as follows: active caspase 3, poly ADP ribose and vimentin (R&D Systems); Akt, Akt-pS473, ErbB2, ErbB2-pY1248, ErK-pT202/T204, FAK-pY925 and Src-pY416 (Cell Signaling); FAK-pY397, FAK-pY576, FAK-pY577 and paxillin-pY31 (Invitrogen); E-cadherin and paxillin (BD Transduction Laboratories); anti-V5 (Serotec); FAK and N-cadherin (Santa Cruz); actin and tubulin (Sigma); FAK pY407, FAK pY861 and paxillin pY118 (Biosource); secondary antibodies (Jackson Laboratory); Hoechst 33528 (Sigma). The FAK inhibitor AZ675 was provided by AstraZeneca (Alderly Edge).

Cancer cell lines were purchased from ATCC, Manassas, VA. Human Mammary Epithelial cells immortalized by telomerase expression (tert-HMEC) have been described previously [35 (link)]. Cells were cultured in Dulbecco's Modified Eagle's Medium supplemented with 10% fetal bovine serum. Cell extracts were prepared as described previously [36 (link)] and immunoblot analysis was carried out with the following antibodies: EGFR (#4267), phospho-EGFR[Y845] (#6963), PARP (#9532), phospho-Akt[T308] (#9275), Akt (#4691), phospho-Erk (#9101), HER2 (#2165), HER3 (#4754), phospho-HER2[Y877] (#2241), and phospho-S6 (#2211) from Cell Signaling Technology, Danvers, MA; Erk (sc-93), phospho-Tyrosine (sc-7020), and Actin (sc-1616) from Santa Cruz Biotechnology, Santa Cruz, CA; phospho-Tyrosine (4G-10, 05–321) from Millipore, Temecula, CA; E-cadherin (610182) from BD Transduction Laboratories, San Jose, CA.

Total cell lysates were prepared in a Tris pH 6.8–10% SDS solution (1:1) by heating at 95° for 20 mins. Protein concentration was measured using Pierce BCA protein assay kit as per the company instructions. For western blotting 10–40 ug of protein was resolved on 7.5% or 10% mini gels from Biorad, transferred to nitrocellulose membrane using semi-dry method and immunoblotted. 10% BSA was used for filter blocking in all conditions.
The following primary antibodies were used: against Notch1, Slug and Actin (Santa Cruz Biotechnology), against PlexinD1 (R&D Systems), against Vinculin (Sigma), against Notch3 (Cell Signaling technology), against E-cadherin (BD Transduction laboratories).