Anti luciferase

Immunohistochemical analysis was performed as described in our previous study. Briefly, all tumor xenograft sections were deparaffinized with xylene and rehydrated through an alcohol gradient, and then, endogenous peroxidase was inactivated by 0.5% H₂O₂ for 10 min. Subsequently, the sections were blocked with 5% normal goat serum for 1 h and washed three times with PBS, followed by incubation at 4 °C overnight with one of the following primary antibodies: anti-5-hmC (1:100, Active Motif), anti-5mC (1:100, Active Motif), anti-TET1 (1:300, Abcam), anti-TET2 (1:300, Abcam), anti-TET3 (1:300, Abcam), anti-DNMT1 (1:100, Abcam), anti-DNMT3A (1:500, Abcam), anti-DNMT3B (1:100, Abcam), anti-Ki 67 (1:200, Abcam), and anti-luciferase (1:1000, Abcam). The sections were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for 1 h and developed with a DAB kit.

Tumor tissues were fixed in 3.7% paraformaldehyde for 24 h and embedded with paraffin, prepared as 4-μm sections, and mounted on slides. Antigens were retrieved by boiling in citrate buffer (DakoCytomation, Glostrup, Denmark) for 5 min. Primary antibodies were incubated overnight at 4°C as follows: anti-luciferase (1:500 dilution; Abcam, Cambridge, UK), anti-HIF-1α (1:100 dilution; Novus Biologicals, Littleton, CO, USA), and anti-Ephrin-A3 (1:500 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Secondary antibodies were used as follows: biotinylated anti-goat for luciferase (1:500 dilution; Dako, Glostrup, Denmark) and biotinylated anti-mouse for HIF-1α and Ephrin-A3 (1:500 dilution; Dako, Glostrup, Denmark). An avidin–biotin peroxidase complex was used to amplify the signal, followed by development using DAB and counterstaining with hematoxylin.

HEK293 cells or macrophages were seeded on poly-L-lysine coverslips 24 h before use. After transfection and/or stimulation, cells were fixed with 4% formaldehyde, blocked using 2% bovine serum albumin (Sigma-Aldrich), and permeabilized with 0.1% Triton X-100 (Sigma-Aldrich). ASC was stained using a primary antibody anti-ASC (polyclonal anti-PYD ASC, Santa Cruz or monoclonal anti-CARD ASC, 1:1,000 dilution; BioLegend) or anti-Luciferase (Abcam; 1:500 dilution) and an Alexa Fluor AF488 or AF647 conjugated secondary antibody (1:200 dilution; Life Technologies). ProLong Diamond Antifade Mountant with DAPI was used as a mounting medium. Images were acquired at room temperature with a Nikon Eclipse Ti microscope equipped with a 20× S Plan Fluor objective (numerical aperture 0.45), a 40×S Plan Fluor objective (numerical aperture 0.6), and a 60×S Plan Apo Vc objective (numerical aperture 1.40), and a digital Sight DS-QiMc camera (Nikon) with a Z optical spacing of 0.2 μm and 387 nm/447 nm, 482 nm/536 nm, 543 nm/593 nm, and 650 nm/668 nm filter sets (Semrock) and NIS Elements software (Nikon). Images were processed using ImageJ software (National Institutes of Health), and the maximum-intensity projection images are shown in the results.

For IHC anti-Cleaved Caspase 3 (Cell signalling #9661), anti-Ki67 (RTU-Lab Vision #RM-9106-R7 Dilution Ready to use), anti-Luciferase (Abcam #ab181640), Mcl-1 (Cell signaling #5453) Ly-6G (GR1), Clone 1A8 (RUO); 551459 BD Pharmigen, F4/80 (BM8) Rat Mono, 14-4801-82 eBioscience^™ (Thermo Scientific), p16 (Abcam #ab211542) were used. For Western blot anti-Cleaved Caspase 3 (Cell signalling #9664), anti-Bcl-2 (Cell signalling #3498 S), anti-Mcl-1 (Cell signalling #5453) anti-HSP90 (Cell signalling #4874), p27 kip1 (Cell signalling #3698 S), p15 ink4b (Abcam #53034), p16 ink4a (Abcam #211542), p21 (Abcam #107099), p19 ARF (5-C3-1) (Santa Cruz Biotechnology #SC-32748), PAI-1 (Abcam #66705), Mcl-1 (Cell signaling #5453), secondary Anti-Rabbit (Promega #W4011), secondary Anti-Mouse (Promega #W4021). For FACS sorting CD326 (EpCAM) Monoclonal Antibody (G8.8), FITC, eBioscience^™ (11-5791-82). Further information about dilutions and clones are available in Source Data.