Integrin β1

Hematoxylin and eosin staining was performed with paraffin sections by Duke Pathology Laboratory (Durham, NC, USA). Immunostaining of OCT-embedded frozen subcutaneous tumor tissue sections were performed as previously described.^{44 (link)} Five-micrometer-thick sections were fixed in 100% methanol for 15 min, blocked with 10% horse serum in PBS (Invitrogen) for 1 h and then incubated with antibodies against Ki-67 (SP6) (Thermo Fisher Scientific, Grand Island, NY, USA); cleaved caspase 3 (#9661), N-cadherin (#13116), E-cadherin (#3195) and β1-Integrin (#9699) (Cell signaling Technology) for 1 h at room temperature or 4 °C overnight. Sections were then washed five times in PBS with 0.05% Tween-20, followed by detection with Alex 555-conjugated secondary antibody (Thermo Fisher Scientific) and counterstaining with 1 μg/ml Hoechst 33258 for 1 min. For immunostaining, paraffin-embedded lung tissue sections were dewaxed in 100% xylene for 5 min followed by sequential 5-min treatments of 100, 90 and 70% ethanol, antigen unmasking by boiling in 10 mM citrate buffer for 10 min and then undergo immunostaining as described above with a primary antibody against Melan-A (HPA048662) (Sigma) and detection with a Dylight 549-conjugated secondary antibody. Fluorescent pictures were taken and processed using the Olympus BX41 microscopic imaging system (Olympus, Center Valley, PA, USA).

Cell monolayers were lysed in lysing buffer containing 1M Tris-HCl (pH 7.5), 3M NaCl, NP₄O, distilled water, a protease inhibitor cocktail (Roche, Switzerland), PMSF and sodium orthovanadate. Cells were centrifuged at 13 000 RPM, 4°C. The amount of protein was measured using the Bradford assay [20 (link)] and stored at -80°C until used. Equal amounts of protein (20 μg) were run on Bolt® Bis-Tris Plus gels (Invitrogen, Thermo Fisher Scientific) and transferred to a nitrocellulose membrane using iBlot® Dry Blotting System (Invitrogen, Thermo Fisher Scientific). The membranes were blocked with 5% skim milk in a TBS buffer with 1% of Tween 20 for 1h and incubated with primary antibodies against vimentin (D21H3) (Cell Signaling Technology, MA, USA) and β1 integrin (Cell Signaling Technology, MA, USA) at 4°C overnight. Membranes were washed 3 times in TBS and incubated with suitable secondary antibodies and then washed 3 times in TBS. Signals were detected using LumiGLO® chemiluminescent substrate (Cell Signalling Technology, Danveers, MA).