Protease inhibitors

First, we lysed the samples digested and collected from tumor tissues of each group with enhanced RIPA Lysis Buffer containing protease inhibitors (Wuhan Boster Biological Technology). The protein concentration was measured using a BCA protein assay kit (Wuhan Boster Biological Technology) to ensure equal protein loading. Subsequently, protein samples were separated via SDS-PAGE and then electro-transferred onto PVDF membranes. On the PVDF membranes, we blocked with 5% BSA at room temperature for 1 h to prevent non-specific binding, then added diluted primary antibodies, including β-actin (ab8226, 1/5000, Abcam), NDUFAF6 (ab110244, 1/1000, Abcam), NRF2 (SAB4501984, 1/1000, Sigma), and PD-L1 (ab213480, 1/1000, Abcam) and incubated overnight at 4 °C for specific protein binding. Afterward, the PVDF membrane was washed three times with PBST for 5 min each, then incubated with Anti-Mouse-HRP secondary antibody (Cat # 7076, 1/5000, CST) or Anti-Rabbit-HRP secondary antibody (Cat # 7074, 1/5000, CST) at room temperature for 1 h. Finally, an appropriate amount of ECL working solution (EMD Millipore, USA) was added to the transferred membrane and incubated at room temperature for 1 min. Excess ECL reagent was removed, sealed with plastic wrap, placed in a dark box, and exposed to X-ray film for 5–10 min, followed by development and fixation.

Cultured cells were detached by trypsin, collected and lysed with enhanced RIPA lysis containing protease inhibitors (Wuhan Boster Biological Technology Co., Ltd., Wuhan, Hubei, China) with the protein concentration measured using BCA protein quantification kit (Boster). Proteins were separated by 10% SDS-PAGE, and the separated proteins were electrotransferred onto a polyvinylidene fluoride membrane and blocked with 5% bovine serum albumin (BSA) for 2 h at ambient temperature to block non-specific binding. The membrane was incubated with diluted primary antibodies (rabbit anti-GAPDH, ab9485, 1: 500, Abcam; rabbit anti-RUNX1, ab272456, 1: 500, Abcam; NF-H, 1: 500, #55453, Cell Signaling Technology [CST], Danvers, MA; NF-M, 1: 500, #84390, CST; NF-L, 1: 500, ab223343, Abcam; β-catenin, 1: 500, ab32572, Abcam) overnight at 4 °C. Then the membrane was then incubated with horseradish peroxidase-labeled goat anti-rabbit secondary antibodies (ab205719; 1: 2000; Abcam) for 1 h, treated with enhanced chemiluminescence working fluid (WBULS0100, EMD Millipore, Billerica, MA) and placed in an optical luminescent instrument (AS4000, GE, Little Chalfont, Buckinghamshire, UK) to develop images. Finally, Image J software was run to quantify the grayscale of each band in the Western blot image normalized to GAPDH.

Cells were collected by trypsin detachment and lysed using Radio-Immunoprecipitation Assay (RIPA) lysis buffer containing the protease inhibitors (Wuhan Boster Biological Technology Ltd., Wuhan, Hubei, China). The concentration of protein was determined using the bicinchoninic acid (BCA) kit (Wuhan Boster Biological Technology Ltd., Wuhan, Hubei, China). The protein was then separated by conducting sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride (PVDF) membrane. A membrane blockade was performed for 1 h and incubated with the following primary rabbit anti-human antibodies to matrix metalloprotein-9 (MMP-9) (ab194316, 1 : 1000), N-cadherin (ab18203, 1 : 1000), Vimentin (ab137321, 1 : 500), E-cadherin (ab15148, 1 : 500), ERK1/2 (ab17942, 1 : 1000), phosphorylated ERK1/2 (ab214362, 1 : 500), MEK1/2 (ab178876, 1 : 5000, Abcam, Cambridge, UK), phosphorylated MEK1/2 (ab194754, 1 : 1000), and GAPDH (ab181602, 1 : 5000) at 4°C overnight. The aforementioned antibodies were provided by Abcam Inc. (Cambridge, UK). The protein bands were developed using the enhanced chemiluminescence reagent and the observations were documented using the SmartView Pro 2000 software (UVCI-2100, Major Science, USA). The Quantity One software analyzed the gray values of protein bands.