Western blot analysis

A549 cells were seeded in a 10 cm² dish and incubated in culture medium for 24 h. Thereafter, the cells were treated with lidocaine (10 µM) or incubated with 20 ng/ml TGF-β (R&D Systems, Inc.) at 37°C for 48 h to induce EMT. The use of human TGF-β to elicit EMT in a mouse lung cancer cell line is also feasible as previously described (25). Subsequently, cells were cultured to logarithmic growth phase, and were collected and lysed in RIPA Lysis and Extraction Buffer (cat. no. R0278-50ML; Sigma-Aldrich; Merck KGaA). The protein concentration of total cell lysates was accessed using the bicinchoninic acid method. A total of 30 µg total proteins/lane were separated by SDS-PAGE on 10% gels (Bio-Rad Laboratories, Inc.), then transferred to a PVDF membrane. The membrane was blocked for 1 h at room temperature in TBST (10 mM Tris, pH 7.5; 150 mM NaCl; 0.1% Tween 20) with 5% skim milk. After incubation with the following primary antibodies for 1 h at room temperature: E-cadherin (1:1,000 dilution; cat. no. 3195), vimentin (1:1,000; cat. no. 5741), Slug (1:1,000; cat. no. 9585) and GAPDH (1:1,000; cat. no. 2118) (all from Cell Signaling Technology, Inc.), the membrane was washed three times in TBST (10 min/wash) and then incubated with secondary antibodies, including anti-rabbit (1:5,000; cat. no. 7074; Cell Signaling Technology, Inc.) and anti-mouse (1:5,000; cat no. 7076; Cell Signaling Technology, Inc.,) in TBST at room temperature for 1 h. The membrane was subsequently washed three times with TBST (10 min/wash) and the bands were visualized using enhanced electrochemiluminescence (ECL pierce kit; cat. no. 32109; Thermo Fisher Scientific, Inc.) to analyze the expression of target proteins. ImageJ software (version 1.48v) was used for semi-quantification of the western blots and all measurements were normalized against the GAPDH loading control.