Anti snail2

Cells were fixed in 4% phosphate-buffered saline (PBS)–paraformaldehyde for 15 min, incubated in 0.1% Triton X-100 for 5 min on ice, then in 0.2% fish skin gelatine in PBS for 1 h and stained for 1 h with an anti-E-cadherin (1:100, BD Biosciences, 610182) or anti-p62/SQSTM1 (1:100, BD Biosciences, 610833) or anti-Snail2 (1:100, Cell Signalling Technology, 9585), p65/RELA (1:100, Cell Signalling Technology, 8242). Protein expression was detected using Alexa Fluor (1:400, Molecular Probes) for 20 min. DAPI or TO-PRO-3 (Invitrogen) was used to stain nuclei (1:1000). Rhodamine phalloidin was used to visualise filamentous actin (F-actin) (Molecular Probes). For immunofluorescence staining of 3D cultures from ATII cells, spheres were fixed with 4% PBS–paraformaldehyde for 40 min, permeabilised in 0.5% Triton X-100 for 10 min on ice and stained with rhodamine phalloidin for 1 h at room temperature. Spheres were counterstained with DAPI. Samples were observed using a confocal microscope system (Leica SP8). Acquired images were analysed using Photoshop (Adobe Systems) according to the guidelines of the journal.

After the transfection for 48 h, whole-cell protein was extracted using a RIPA lysis buffer. The protein concentrations were detected by bicinchoninic acid method. Then, the protein extracts were separated using SDS-PAGE and transferred to a polyvinylidene fluoride membrane. After blocking with skimmed milk, the protein was incubated with the primary antibodies for 1 h, followed by secondary antibodies for 1 h. Primary antibodies, including anti-Bcl-2, anti-Bax, anti-total Caspase 3, anti-cleaved Caspase 3, anti-GAPDH, anti-Vimentin, anti-Cyclin D1, anti-Snail2, anti-p-AKT (Ser473), anti-p-mTOR (Ser2448), and anti-p70S6K (1:1,000), were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA).

Immunoblotting was carried out according to standard procedures.^{12 (link)} The following primary antibodies were used in this study: anti-EGFP (ImmunoKontact, UK; catalog number 210-PS-1GFP), anti-ZEB1 (Santa Cruz Biotechnology, Dallas, TX, USA; sc-25388); anti-SNAIL2 and anti-Cyclin D1 (Cell Signaling Technology, Danvers, USA; 9585 and 2978) anti-ZEB2 (in-house^{12 (link)}); anti-HA and anti-α-Tubulin antibodies from Sigma-Aldrich (St Louis, MO, USA; H3663 and T5168); anti-TWIST1 antibody from Abcam (Cambridge, MA, USA; AB50887); and anti-E-cadherin (BD Bioscience, San Jose, USA; 610181).