Dylight650

The following antibodies against human proteins were employed unless otherwise noted: goat (M20) and rabbit (M185) polyclonal anti‐mouse PECAM‐1 antibodies and anti‐GAPDH antibody from Santa Cruz Biotechnology (Santa Cruz, CA); 390, rat anti‐mouse PECAM‐1 antibody (DeLisser et al. 1997), MEC 13.3, rat anti‐mouse PECAM‐1 (DeLisser et al. 1997) and DyLight650 conjugated antibody from Novus Biologicals (Littleton CO); anti‐mouse CD31, Alexa 647 conjugated antibody from Southern Biotech (Birmingham, AL); 390, MEC 13.3 and rat IgG2a, κ isotype control from BioLegend (San Diego, CA); donkey anti‐goat IgG, goat anti‐mouse alexa594 conjugated from Life Technologies (Grand Island, NY); anti‐paxillin antibody (BD Transduction Laboratories (Lexington, KY); antiphosphotyrosine antibody and HRP‐conjugated, goat anti‐mouse antibody from EMD Millipore (Billerica, MA); and anti‐EGFR and anti‐Cdc42 antibodies from Cell Signaling Technology (Danvers, MA).

For tumors, single cell suspensions were generated using a mouse tumor dissociation kit in combination with the gentleMACS^™ Octo Desiccator (Miltenyi Biotec, Auburn, CA) per manufacturer protocol. Cell lines were harvested with a cell stripper dissociation reagent (Corning, Manassa, VA). Single cell solutions were washed twice in PBS and resuspended in 1X FACS buffer at 1 × 10⁷ cells/ml. Prior to staining, the monoclonal antibody 7.16.4 and IgG2a isotype control were first conjugated to DyLight^™ 650 using the Lightning-Link antibody labeling kit (Novus Biologicals, Centennial, CO). Antibodies were added at 0.5 ug per 100 μl of cell sample and incubated for 30 min on ice, alongside a set of unstained samples. Samples were washed 4X with FACS buffer with centrifugations at 400G @ 5 min each. After the final wash, samples were resuspended in 0.5 ml FACS buffer with the viability dye SYTOX Green (1:1000), except for selected controls. Fluorescence was detected with a Attune NxT Cytometer (Thermo Fisher Scientific, Carlsbad, CA) and gated on viability. Plots and calculations were analyzed with the FCS Express software.

The lungs were embedded in paraffin blocks, sliced into 10μm-thick sections, deparaffinized, and subjected to antigen retrieval. The slides were blocked with 10% BSA for 15 min at room temperature and incubated with primary antibodies at a 1:50 dilution for 1 hour in the dark. The antibodies used were FABP4 (NOVUS AF1443), CD31 (ThermoFisher PA5–32321), CLIC4 (NOVUS NBP1–00172), SCN7A (ThermoFisher BS-12127R) and Apelin (ThermoFisher APEL-FITC). On the following day, slides were probed for 1 hour with a secondary antibodies Donkey anti-Goat Secondary Antibody PE (Novus NB7590), Donkey anti-Mouse Secondary Antibody Alexa Fluor^™ 488 (Thermo A-21202), Donkey anti-Goat Secondary Antibody PE (Novus NB7590), and Donkey anti-Rabbit Secondary Antibody DyLight 650 (Novus NBP1–75644). The slides were washed in TBST and water followed by addition of a drop of ProLong^™ Diamond Antifade Mountant with DAPI (ThermoFisher P36962). To obtain confocal and 3D images, microscopy was performed using the Zeiss LSM880 confocal microscope with Airyscan at 60X oil immersion and accompanying ZenBlack software (version 2.3, Carl Zeiss Microscopy), and the resulting z-stacked images were processed using imageJ (NIH) and into 3D renders using Imaris software (version 534 10.0, Oxford Instruments).