Ripa lysis

Total protein was extracted using RIPA lysis (Sigma, St. Louis, MO). Mitochondrial and cytosolic proteins were collected using Mitochondria/Cytosol Fractionation Kit (EMD Millipore, Massachusetts, USA) referring to manufacturer’s protocol. Cell lysates (20 μg) fractionated electrophoretically by 12% SDS-PAGE gels, and transferred onto PVDF membranes (GE Healthcare, Freiburg, DE). Primary antibodies against Bax, Bcl-2, Caspase-9, Cyt C, PTEN, p-Akt (Ser473), Akt, p-GSK3β (Ser9), GSK3β, β-catenin and β-actin (Cell Signaling Technology, Danvers, MA) were probed with proteins on the membrane at 4 °C overnight. After incubating with secondary antibodies (Cell Signaling Technology, Danvers, MA), the results were detected by ECL Advance reagent (GE Healthcare, Freiburg, DE) with ChemiDoc XRS System and analyzed by Image Lab Software (Bio-Rad, Kidlington, UK).

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).

Total proteins from spinal cord tissue or bEnd.3 cells were extracted using RIPA lysis (Sigma, United States) and protease inhibitor (Sigma, United States). The tissue or cells were fully lysed after ultrasonic treatment for 120 s. Subsequently, the lysate was centrifuged at 4 °C and 12 000  r/min for 15 min, and the supernatant was collected. The protein concentration was determined using the BCA protein detection kit and a microplate reader (Thermo Fisher, United States). A 10% gel was utilized to separate proteins with different molecular weights. After adding samples and markers, the voltage was adjusted to 90 V for electrophoresis until the protein bands were completely separated. The proteins were then electro-transferred to a polyvinylidene fluoride (PVDF) membrane at 300 mA for 90 min. The PVDF membrane was blocked with 5% non-fat milk for 1.5 h and incubated with corresponding primary antibodies at 4 °C overnight. On the following day, the primary antibodies were removed, and the membrane was rinsed with TBST solution three times. Subsequently, secondary antibodies were added for incubation at room temperature for 1 h. Bands were covered with ECL chemiluminescence (Millipore, United States) and visualized using a chemiluminescence instrument (Bio-Rad, United States). Actin was used as an internal reference. Antibodies used are listed in Table S1.