Ab92536

The tissue is fixed in formalin and embedded in paraffin, and then cut into thin slices. The sections were deparaffinized with xylene and hydrated with ethanol, and then the antigen was recovered by pressure cooking. At room temperature, the sections were sliced with TNC primary antibody (ab108930, Abcam, Cambridge, MA), MMP9 (Abcam, ab76003, 1:1000 dilution), MMP2 (Abcam, ab92536, 1:1000 dilution) and CD31 (ab134168, Abcam), incubated for 60 min and then incubated with IgG H&L (HRP; 1:200 dilution, Abcam, Cambridge, UK). Then, the sections were stained with chromogen and counterstained with hematoxylin. The score is based on the intensity of staining (0 means no staining, 1 means weak staining, 2 means moderate staining, and 3 means strong staining). The product of the two levels was calculated as the final expression score.

The primary antibodies used were anti-HuR antibody (11910-1-AP, Proteintech), anti-PTBP1 antibody (12582-1-AP, Proteintech), anti-RHOBTB3 antibody (13945-1-AP, Proteintech), anti-E-cadherin antibody (3195, Cell Signaling Technology), anti-N-cadherin antibody (sc-8424, Santa Cruz), anti-CD44 antibody (15675-1-AP, Proteintech), anti-MMP2 antibody (ab92536, Abcam), anti-ADAR3 antibody (sc-73410, Santa Cruz), anti-PKM1 antibody (7067, Cell Signaling Technology), anti-PKM2 antibody (4053, Cell Signaling Technology), and anti-β-actin antibody (3700, Cell Signaling Technology). CRC cells were collected, washed and lysed in RIPA lysis buffer (Beyotime, Hangzhou, China). Then, the protein concentration was determined using a BCA Protein Assay Kit (Beyotime). Cell lysates were separated by 8-12% SDS-PAGE and then transfected into PVDF membranes (Millipore, Schwalbach, Germany). The membrane was blocked with TBST buffer containing 5% skim milk powder and incubated with the corresponding primary antibodies at 4 °C overnight. The membrane was hybridized with an HRP-conjugated secondary antibody (FDM007 and FDR007, Fudebio, Hangzhou, China) at room temperature for 1 h. The signals were detected using an enhanced chemiluminescence kit (FD8030, Fudebio, Hangzhou, China).

Thymoquinone (TQ) was product of MCE (CAS: 490-91-5). Hoechst 33342 (B2261) was bought from Sigma-Aldrich. Cell counting kit‐8 (CCK‐8) was product of Selleck Chemicals (US). N-Acetyl-L-cysteine (NAC) and Z‐VAD‐FMK (ZVF) were obtained from Beyotime Biotechnology (Shanghai, China). The Apoptosis Detection Kit #556547 was purchased from BD (San Jose, CA). Antibodies against Bax (ab32503), Bcl‐2 (ab182858), LC3B (ab192890), Beclin-1 (ab207612), ATG7 (ab133528), MMP2 (ab92536), MMP9 (ab76003) and PD-L1 (ab213524) were bought from Abcam (San Francisco, CA). Antibody against vimentin (V6389) was purchased from Sigma-Aldrich (US). Antibodies against β‐actin (20536‐1‐AP) and Bcl-xl (10783-1-AP) were obtained from Proteintech (Chicago, IL). Antibodies against cleaved caspase 3 (9664), cleaved poly (ADP‐ribose) polymerase (PARP) (5625), E-cadherin (3195), N-cadherin (13116), and SQSTM1/p62 (8025) were obtained from CST company (NJ, US).

The cell lysates were collected, and 30 mg protein of each sample was separated using 10% SDS-PAGE. The protein was then electrotransferred onto Polyvinylidene Fluoride (PVDF) membrane (Millipore, Boston, MA, USA). The PVDF membrane was sealed in 10% skimmed milk. Then the membrane was incubated with primary antibodies, including anti-TNC (Abcam, ab108930, 1:1000 dilution), anti-GAPDH (Abcam, ab181602, 1:10,000 dilution), and anti-ERK (Abcam, ab184699, 1:1000 dilution)), anti-p-ERK (Abcam, ab201015, 1:1000 dilution), anti-MMP9 (Abcam, ab76003, 1:1000 dilution), anti-MMP2 (Abcam, ab92536, 1:1000 dilution), anti-E-cadherin (Abcam, ab1416, 1:1000 dilution), anti-N-cadherin (Abcam, ab18203, 1:1000 dilution), anti-vimentin (Abcam, ab92547, 1:1000 dilution), anti-snail (Abcam, ab216347, 1:1000 dilution), anti-clumping (Abcam, ab27568, 1:1000 dilution) and anti-TWIST1 (Abcam, ab50887, 1:1000 dilution) overnight. Then the membrane and horseradish peroxidase-conjugated secondary antibody (Abcam AB6721, 1:10,000 dilution) were incubated for 2 h at room temperature. Chemiluminescence detection reagent (Millipore, Boston, MA, USA) was used to visualize protein bands.

The protein level of TRIM16 was detected using Western blot, according to the experimental procedures recorded in the previous publications [29 (link)]. Briefly, transfected cells were lysed in Radioimmunoprecipitation assay (RIPA) lysis buffer with protease inhibitor (Thermo Fisher Scientific) to extract total protein. The protein quantification was measured by the BCA^TM Protein Assay Kit (Pierce, Appleton, WI, USA). Then the equivalent protein (50 µg) was added to the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to obtain the target protein. Next, the target proteins in the gel were transferred onto polyvinylidene difluoride (PVDF) membranes and incubated with primary antibodies against TRIM16 (ab72129, 1:2000, Abcam, Cambridge, MA, USA), hexokinase 2 (HK2, ab227198, 1:5000, Abcam), B-cell lymphoma protein 2 (Bcl-2)-associated X (BAX, ab32503, 1:2000, Abcam), Matrix metalloproteinase 2 (MMP2, ab92536, 1:1000, Abcam), Proliferating cell nuclear antigen (PCNA, ab18197, 1:1000, Abcam), and β-actin at 4°C overnight. The membranes were then incubated with second antibodies (Horseradish peroxidase-conjugated IgG antibody). Finally, the Western blot intensities were detected using LAS 4000 Image Reader (Fujifilm, Tokyo, Japan)

Total proteins from cells and tissues were extracted by the RIPA lysate. Protein quantification was conducted with a BCA protein quantification kit. An appropriate amount of protein sample was denatured at 100°C for 3–5 min and then subjected to protein electrophoresis using 10% SDS-PAGE for 1 h. the samples were transferred to PVDF membranes. The membranes were blocked in Tris Buffered Saline with Tween 20 blocking solution containing 5% skim milk powder at room temperature for 2 h. The membranes were washed three times with TBST and incubated overnight at 4°C using primary antibodies against MMP-2, MMP-9, p-PI3K, PI3K, p-AKT, AKT, and GAPDH (ab92536, ab76003, ab278545, ab140307, ab192623, ab8805, ab8245; all from Abcam, Shanghai, China). The next day, the horseradish peroxidase-labeled goat anti-rabbit secondary antibody IgG (ab96899) and goat anti-mouse secondary antibody IgG (ab96879) dilution buffer were added, and membranes were incubated with these secondary antibodies at room temperature for 2 h. The enhanced chemiluminescence reagent was added dropwise, and the membranes were imaged by the gel imaging system. The gray value of each band was analyzed by the ImageJ software. The results were expressed as the ratio of the gray value of the target protein to GAPDH, the internal reference protein.