Lumi light

Tert-butyl hydroperoxide (t-BHP), sodium dodecyl sulfate (SDS), phosphotungstic acid, buthylated hydroxytoluene, 2-thiobarbituric acid and malonaldehyde bis(dimethyl acetal), the Bicinchoninic protein determination assay and the anti-α-tubulin were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). The following were purchased from Abcam Inc., (Cambridge, MA, USA): rabbit polyclonal anti-PRDX1, mouse monoclonal anti-PRDX6, mouse monoclonal anti-4-Hydroxynonenal (4-HNE), rabbit polyclonal anti-catalase, and 8-hydroxy-deoxyguanosine (8-OHdG). Anti-thioredoxin 1 antibody was purchased from Cell Singaling Thecnology (Danvers, MA, USA), Polyvinylidene fluoride (PVDF) membranes (0.22 µm pore size; Osmonics Inc., Minnetonka, MN, USA), donkey anti-rabbit IgG and goat anti-mouse IgG, both conjugated to horseradish peroxidase (Cedarlane Laboratories Ltd., Hornby, ON, Canada), an enhanced chemiluminescence kit (Lumi-Light; Roche Molecular Biochemicals, Laval, QC, Canada) and radiographic films (Denville Scientific, Inc., Saint-Laurent, QC, Canada) were also used for immunodetection of blotted proteins. Other chemicals were used at the reagent level.

Cultured cells were lysed in Mammalian Protein Extraction Reagent (MPER, Thermo Scientific), supplemented with protease inhibitor and phosphatase inhibitor cocktail (Thermo Scientific). Proteins were separated on SDS-polyacrylamide gels (SDS-PAGE) and transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore). Membranes were blocked in 5% milk or bovine serum albumin (BSA) in Tris-buffered saline, with 0.05% Tween-20. Immunodetection was done with antibodies directed against BRCA2 (1:1000, Calbiochem, #OP95), BRCA1 (1:1000, Cell Signaling, #9010), cGAS (1:1000, Cell Signaling, #15102), STING (1:1000, Cell Signaling, #13647), cMYC (1:200, Santa Cruz, sc40, Abcam (ab32072) 1:1000), pIRF3 (1:1000, Cell Signaling, # 29047), IRF3 (1:1000, Cell Signaling, # 4302), STAT1 (1:1000, Cell Signaling, # 9172), pSTAT1 (1:1000, Cell Signaling, # 8826) and beta-Actin (1:10.000, MP Biochemicals, #69100). Horseradish peroxidase-conjugated secondary antibodies (1:2500, DAKO) were used and visualized with chemiluminescence (Lumi-Light, Roche diagnostics) on a Bio-Rad Bioluminescence device equipped with Quantity One/Chemidoc XRS software (Bio-Rad).

The details of antibodies used for flow cytometry and Western blotting are provided in Supplementary material, Table S1. Western blotting detection was performed using Lumi‐Light Western blotting substrate (Roche Diagnostics, Basel, Switzerland). Cells were treated under normal culture conditions with EGF, HGF, IFNγ (each from R&D Systems, Minneapolis, MN, USA), erlotinib (LC Laboratories, Woburn, MA, USA), cetuximab (Merck KGaA Darmstadt, Germany), selumetinib (AZD6244, Axon Medchem, Reston, VA, USA), XL147 (LC Laboratories), everolimus (Selleckchem, Houston, TX, USA), BMS911543 (Selleckchem) and actinomycin D (Sigma‐Aldrich).

After SDS-PAGE, proteins were transferred by semi-dry blotting to polyvinylidene difluoride membranes (Millipore, Burlington, VT), blocked for 90 minutes in 5% non-fat milk/TBST and probed overnight at 4°C with the respective primary antibody (list of antibodies and dilutions: see Table S1) in 5% non-fat milk/TBST. Immune complexes were detected with HRP-conjugated secondary antibodies (Table S1) and visualized using enhanced chemiluminescence detection reagent (Lumi-light, Roche Diagnostics, Rotkreuz, Switzerland) and ImageQuant LAS 4000 (GE Healthcare, Chicago, IL). Protein bands were quantified using ImageJ 1.50i (Wayne Rasband, National Institutes of Health, Bethesda, MD). Results were presented as fold changes of sham transduced H69 cells, normalized per experiment.

Recombinant SaPLIγ was expressed in 6-histidine tag form by pET28c vector in our laboratory [28 (link)]. The His6-PLIγ and natural saPLIγ were separated under reducing conditions on 12% SDS-PAGE gel, and transferred to different PVDF membranes. Immunodetection was performed by incubating the membranes overnight at 4 °C, stirring, with the PLIγ mAbs at a dilution of 1:500. The reaction was developed using goat anti-mouse peroxidase IgG at a dilution of 1:10,000 and the chemiluminescent substrate Lumi-light (Roche). Images were captured with ChemiDoc XRS⁺ system (Bio-Rad). Parallel Western blot using anti-His6 antibody was conducted to detect His6-PLIγ fusion protein and validate the specificity of PLIγ mAbs.