*5.6. Western Blotting*

Spheroids of MRC-5/A459 were lysed, and total cellular protein was extracted using RIPA lysis (Sigma Aldrich, R0278) with a protease and phosphatase cocktail (Sigma Aldrich, P0044; P5726; P8340). Cell lysates were then centrifuged at 12,000 rpm for 30 min at 4 ◦C, the supernatant containing the soluble proteins was collected and measured by the BCA protein assay (Novagen®, 71285). The samples containing 30 μg protein were subjected to SDS/PAGE under reducing conditions on a 4–20% gradient polyacrylamide gel (Bio-Rad, cat no. 456–1094, Hercules, CA, USA). Following electrophoresis, proteins were transferred to a nitrocellulose membrane, which was then blocked with TBS-T (20 mM Tris-HCl, 150 mM NaCl, and 0.1% Tween 20) containing 5% nonfat dry milk for 2 h. The membranes were incubated with primary antibodies: anti-E-cadherin (1:1000), anti-N-cadherin (1:1000), anti-vimentin (1:5000), anti-α-SMA (1:2000), anti-integrin αv (1:2000), and anti-GAPDH (1:1000) overnight at 4 ◦C (Abcam). The membrane was subsequently incubated with

peroxidase-conjugated secondary antibody (anti-mouse IgG and anti-goat IgG) for 2 h. Detection was performed with an enhanced chemiluminescence kit (Thermo Scientific, Rockford, IL, USA). The signals were detected with an image acquisition system (Alliance 9.7, Uvitec, Cambridge, UK). Band intensities were measured with Image J software (NIH).
