Nf κb1

Cells were seeded on glass coverslips, washed three times with PBS, fixed for 10 min with 4% PFA at 37°C, and then cells were washed three times with PBS and permeabilized for 1 h in block solution (1% BSA, 22.52 mg/mL glycine and 0.1% Tween 20 in PBS). Primary antibodies (TOM20 Polyclonal antibody, 11802‐1, and TOM20 Monoclonal antibody, 66777‐1, 1:200; Proteintech, China; cGAS (1:200, Affinity, China); dsDNA, 1:60, Merck; IκBα (1:100, Cell Signalling Technology); and NF‐κB1 (1:100, Abcam) were diluted in block solution and incubated with cells overnight at room temperature. The cells were washed three times with PBS and incubated with secondary antibodies (anti‐mouse Alexa 488; anti‐rabbit Alexa 594, Proteintech, China, 1:200) for 1 h in the dark. Finally, the cells were incubated with 1 μg/mL DAPI for 15 min and then washed three times with PBS. Images were acquired using a laser scanning confocal microscope (LeicaSP8, Germany).

The sections were deparaffinized in xylene and rehydrated in graded alcohols. Following deparaffinization, antigen retrieval was performed by immersing the sections in 10 mmol citrate buffer (pH 6.0) and heating them twice in a microwave oven (95°C) for 5 min. The sections were incubated with primary antibodies against NFKBIA (Clone ID: EP697; Epitomics; dilution 1:100 overnight at 4°C) and NF-κB1 (Abcam, Tokyo, Japan). The sections were subsequently incubated using the Cell and Tissue Staining kit HRP-DAB system (R&D Systems), according to the manufacturer’s instructions. Immunostainings were performed with known positive and negative tumor controls, and were blindly evaluated by a pathologist.