Af6032

Preprocessed samples were added to the RIPA lysate solution (P0013B; Beyotime, China), and the supernatants were collected after centrifugation. Samples were loaded onto sodium dodecyl sulfate-polyacrylamide gels (3250GR500; neoFROXX, Einhausen, Germany). Proteins were transferred to polyvinylidene difluoride membranes (WGPVDF22; Servicebio, China) after electrophoresis in transfer buffer for 5 min. The membranes were then incubated with primary antibodies (1:5000 dilution) targeting CD31 (AF6191, Affinity, USA), Vimentin (BF8006, Affinity), α-SMA (AF1032, Affinity), slug (350,136, ZENBIO, China), snail (AF6032, Affinity), twist (AF4009, Affinity), LC3II (3868S, CST, USA), p62 (AF5384, Affinity), beclin 1 (AF5128, Affinity), AMPK (AF6423, Affinity), p-AMPK (AF3423, Affinity), mTOR (AF6308, Affinity), and p-mTOR (AF3308, Affinity). Horseradish peroxidase-conjugated secondary antibodies at a 1:10,000 dilution were added after washing. The membranes were then viewed with an automatic darkroom exposure instrument (JS-M6P; P&Q, China) and varying luminous intensities were used for optimal exposure.

At 24 h following transfection, the cells cultured on glass coverslips were fixed in 4% paraformaldehyde for 15 min, permeabilized with 0.01% Triton X-100 for 20 min at room temperature, blocked with 5% goat serum for 1 h at 37°C, and incubated with primary antibodies against non-phospho (active) β-catenin (CST, 19,807, 1:100) and Snail1 (Affinity, AF6032, 1:100) overnight at 4°C. The secondary antibodies were Alexa 488-conjugated goat anti-mouse or anti-rabbit IgG (Abcam). 4′,6-diamidino-2-phenylindole (10 µg/mL, 32,670; Sigma-Aldrich) was used to counterstain the nuclei. Fluorescent images were obtained under a microscope (Carl Zeiss, Germany) and processed using Photoshop software (Adobe).

Protein extraction and Western blot were performed as previously described.12 (link) Antibodies targeting GAPDH (Abcam, ab8245, 1:1000), β-actin (CST, 4970, 1:1000), TP531NP2 (Biovision, A1169-100, 1:500), Vimentin (CST, 5741, 1:1000), E-cadherin (CST, 3195, 1:1000), N-cadherin (CST, 13,116, 1:1000), β-catenin (CST, 8480, 1:1000), non-phospho (active) β-catenin (CST, 19,807, 1:1000), Snail1 (Affinity, AF6032, 1:500), and Histone H3 (Affinity, AF0863, 1:500) were used with the secondary antibody horseradish peroxidase-labeled goat antirabbit (CST, 7074, 1:4000) or anti-mouse (CST, 7076, 1:4000) and incubated at room temperature for 1 h. The protein bands were visualized using enhanced chemiluminescence.