Ecl substrate

Protein was extracted from the cells(CAF, A549 and H1975 cell) by lysis buffer, separated electrophoretically by 10% SDS-polyacrylamide gel and transferred to a PVDF membrane (Millipore, USA). The membranes were blocked for 2 hours with 5% non-fat milk at room temperature, incubated with first antibody to α-SMA antibody (1:1000, Abcam, USA), E-cadherin (1:1000, Abcam, USA), STAT3 antibody (1:1000, arigobio, China), p-STAT3(Tyr705) antibody (1:1000, Millipore, USA), BCL-2 antibody (1:500, Abcam, USA), Survivin antibody (1:500, Abcam, USA) and GAPDH (1:2000, Abcam, USA) overnight at 4 °C and then incubated with second antibody for two hours at 37 °C. Proteins were then displayed with ECL substrate (Cell Signaling Technology, USA) in accordance with the manufacturer’s instructions. Protein levels were normalized to GAPDH levels.

Protein lysates were prepared from T-cell pellets using RIPA (Sigma-Aldrich) solution and the Halt™ Protease/Phosphatase Inhibitor Single-Use Cocktail (Thermo Fisher Scientific). Protein concentrations were determined using a BCA protein assay Kit (Thermo Fisher Scientific). Proteins (>10 µg) were separated on a sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) system and transferred by wet blotting (Biorad, Hercules, California, USA). Western blots were probed using primary antibodies against Tubulin, CPT1a, Bcl-2, Bcl-xL, and Mcl-1 (Cell Signaling Technology Inc., Danvers, Massachusetts, USA and Supplementary Table 3). Bands were detected using horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgGs (Cell Signaling Technology; Agilent) and an ECL substrate (Cell Signaling Technology Inc.).

The proteins derived from 4T1 tumor tissues were extracted and further quantified by the BCA kit. Subsequently, the samples with equal amounts of proteins (20 μg) were separated through SDS-PAGE gel. Then the PVDF membranes with transferred proteins were blocked by 5% skim milk. After incubation with primary antibodies, including phospho-IRF-3 (Ser396) (4D4G) (4947 S, Cell Signaling Technology, 1:1000 dilution), IRF-3 (D83B9) (4302 S, Cell Signaling Technology, 1:1000 dilution), phospho-TBK1/NAK (Ser172) (D52C2) (5483 S, Cell Signaling Technology, 1:1000 dilution), TBK1/NAK (D1B4) (3504 T, Cell Signaling Technology, 1:1000 dilution), phospho-STING (Ser365) (D8F4W) (72971 S, Cell Signaling Technology, 1:1000 dilution), STING (A21051, ABclonal, 1:10000 dilution), and Tubulin β (bs-20694R, Bioss, 1:1000 dilution) overnight at 4 °C, the PVDF films were incubated with anti-rabbit IgG, HRP-linked antibody (7074 S, Cell Signaling Technology, 1:2000 dilution) for another 1 h. Chemiluminescence detection was carried out for protein band visualization with ECL Substrate. Uncropped and unprocessed full scan images of all western blots can be found in the Source Data file.