Patient samples and cell lines

In this study, NSCLC samples and adjacent noncancerous tissues were collected from 10 patients. The Ethical Committee of Jiangmen Central Hospital approved the study, and all patients provided informed consent. Four NSCLC cell lines (A549, H460, H1650, and sk-lu-1) and a human normal bronchial epithelial cell line (BEAS-2B) were cultured in Dulbecco’s modified Eagle medium (DMEM, Gibco, Carlsbad, USA). The chemical reagents MG-132 and cycloheximide (CHX) were obtained from SelleckChem (Houston, USA), and MI-2 was obtained from TargetMol (Boston, USA).

Immunohistochemistry

The NSCLC sections were stained with a MALT1 antibody (Proteintech, 66225–1-Ig, Wuhan, China), and the staining intensity was rated from negative to strongly positive on a scale of 0 ~ 3. A score of 0 ~ 4 corresponds to the percentage of positive cells as no positive cells, less than 10%, 10–50%, 50–80%, and greater than 80%, respectively. The product of these two scores was the final score. The scoring was performed by a pathologist double-blinded to the antigen probe and sample identity.

Western blotting and co-immunoprecipitation

Protein was extracted in cell lysis buffer (Beyotime, Shanghai, China) containing protease inhibitor (SelleckChem), separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and blotted onto PVDF membranes (Millipore, MA, USA), which incubated with primary antibodies against MALT1 (CST, 2494, Danvers, USA), CSN5 (Proteintech, 27511–1-AP), FBXO3 (Proteintech, 17803–1-AP), p-p65 (Absci, AB11014, OR, USA), Ub (Proteintech, 10201–2-AP), β-actin (Santa Cruz, 47778, CA, USA), and GAPDH (Santa Cruz, 47724). The bound primary antibodies were visualized using Luminata Forte Western HRP substrate (Millipore) after incubation with secondary antibody anti-mouse IgG-HRP or anti-rabbit IgG-HRP (Invitrogen, CA, USA). Co-immunoprecipitation (Co-IP) was conducted by using protein A/G magnetic beads (Bimake, Houston, USA) following the manufacturer’s instructions.

Reverse transcription and qPCR

TRIzol (Invitrogen) was utilized to extract total RNA following instructions of manufacturer. Evo M-MLV RT Kit (Accurate Biology, Hunan, China) and PerfectStart Green qPCR SuperMix (TransGen Biotech, Beijing, China) were utilized for reverse transcription and qPCR, respectively. GAPDH was used as an internal reference gene and qPCR was conducted on LightCycler 96 Detection System (Roche, Basel, Switzerland). The primer sequences are shown in Table S1.

Cell proliferation assay

A Cell Counting Kit-8 (CCK-8, Dojindo, Kumamoto, Japan) measured cell viability. Cells were transfected with siRNA (RIBOBIO, Guangzhou, China) or plasmid (Hunan Fenghui Biotechnology Co., Hunan, China), and the siRNA sequences are shown in Table S1. Optical density (OD) values were detected at 0, 24, 48, and 72 h after transfection. Cells were seeded into 6-well plates (1,000 cells/well) for colony formation assay and incubated for 2 weeks. Then cells were fixed in 3.7% formaldehyde for 30 min and stained with 0.1% crystal violet. ImageJ was used to count colonies (≥ 50 cells).

For radiation sensitivity assay, cells were irradiated at varying doses (0, 2, 4, 6, 8 Gy) using the Faxitron MultiRad 225 X-ray machine (2 Gy/min, MA, USA). The surviving fraction was calculated as follows: colonies counted/ (cells plated × plating efficiency), where plating efficiency calculated as follows: (unirradiated colony counts/cells seeded for unirradiated controls) × 100%.

Migration and invasion assays

After transfection of siRNA or plasmid, the migration of cells was evaluated by seeding them into 8 μm cell culture inserts of 24-well plates (Millipore). Matrigel-coated chambers (Corning, NY, USA) were utilized for cell invasion assay. After a culture period of 14-16 h, the cells in upper chambers were fixed via 3.7% formaldehyde and permeabilized with methanol. Then, 0.1% crystal violet stained the upper chambers and the images were obtained by microscope (ZEISS, Oberkochen, Germany).

Dual-luciferase reporter assay

Cells were co-transfected with pNFκB-luc plasmid (Beyotime) and siRNA or plasmid; after 48 h incubation, luciferase intensity was measured by a dual-luciferase reporter assay kit (Beyotime) following the manufacturer’s instructions.

LC–MS analysis

The immunoprecipitated protein mix was subjected to SDS-PAGE and digested by In-Gel Tryptic Digestion Kit (Thermo Scientific, MA, USA). The resulting digest was lyophilized, dissolved in a 1% formic acid solution, and subsequently analyzed using a Thermo Fisher Scientific Orbitrap Fusion LC–MS in positive ion mode. The data were searched using Proteome Discoverer software (Thermo Scientific).

Immunofluorescence assay

A 12-well plate was seeded with cells on glass coverslips for 24 h. Following treatment, the cells were fixed and permeabilized with 4% paraformaldehyde and 0.5% NP-40, respectively. Then, cells were blocked with 3% BSA and incubated with primary antibodies and specific fluorescence-conjugated secondary antibodies. The primary antibodies were used as follows: Rad51 (Abcam, ab133534, Cambridge, UK), phosphorylated DNA-PKcs (Abcam, ab124918), and γ-H2AX (Abcam, ab26350). DAPI was used to stain the nuclei. The ZEISS confocal laser scanning microscope was utilized to acquire images.

Animals

Athymic nude mice (male, 6–8 weeks of age) were obtained from Guangdong Medical Laboratory Animal Center. Southern Medical University Animal Care and Use Committee approved all animal experiments. For the subcutaneous xenograft model, siNC- or siMALT1- transfected H460 cells (5 × 106) were subcutaneously injected into the two flanks of nude mice. SiNC and siMALT1 were injected into the corresponding tumors every 2 days, beginning 7 days after cell injection. Tumor volume was calculated as longest diameter × (shortest diameter)2/2 and measured every 2 days.

For the orthotopic lung cancer model, mice were anesthetized and positioned in the right lateral decubitus. Luciferase-tagged A549 cells (4 × 106) were directly injected into the left lateral thorax (3-5 mm depth) using an insulin syringe. Tumor growth was monitored every 7 days using the noninvasive bioluminescence imaging system (Bruker FX Pro, MA, USA). Mice were randomized into two groups after 1 week injection and treated daily with DMSO or MI-2 (25 mg/kg) via intraperitoneal injection.

Statistical analysis

The experimental results were expressed as mean ± standard deviation (SD). One-way ANOVA was performed to determine the significance of difference between two groups using SPSS 22.0 software. P-value less than 0.05 were considered significant.