M1168

C57BL/6 mouse primary spleen endothelial cells (pMSECs, C57-6057, Cell Biologics) were cultured according to the instructions of the supplier in complete mouse endothelial cell medium (M1168, Cell Biologics) in a humidified 5% (v/v) CO₂ incubator at 37°C. 50% confluent pMSECs were transduced with lentiviral vectors in the presence of 4 μg/ml polybrene (Hexadimethrine bromide, 107689, Sigma-Aldrich). Puromycin (ANT-PR-1, InvivoGen) was added to cultures 48 h after infection and drug selection continued until all control cells were dead. pMSECs were seeded in triplicates at density of 2,000 cells per well in 96-well plates and analysed after 1, 3, 5 and 7 days using the SRB assay [48 (link)]. For allograft assays, 1 × 10⁶ pMSECs were injected subcutaneously into SCID/beige mice.

Primary brain microvascular endothelial cells from C57 black 6 mice (C57BL/6‐mBMEC, #C57‐ 6023) and complete mouse endothelial cell medium (M1168) were acquired from Cell Biologics. Quantikine ELISA kits were obtained from R & D systems. Subcellular Protein Fractionation and Pierce BCA Protein Assay Kits (respectively #78840 and #23225) were purchased from Thermo Scientific. anti‐claudin‐5 (#352500) and rabbit anti‐Nrf2 (#PA5‐88084) were purchased from Invitrogen; the rest of the antibodies were purchased from other sources mentioned in our previous publications.⁵, ²⁰

hRECs (PB-CH-160-8511, Pelo Bioscience) were maintained in EGM-2 MV medium (CC-3202, Lonza) under treatment conditions for 2 weeks. In addition to the 2 treatment conditions with 1000 U IFN-β (8499-IF-010, R&D Systems) and 33 μg/mL cGAMP (tlrl-nacga23m, InvivoGen), a control arm was performed. Meanwhile, the cells underwent 3 passages (p7–p9) and the medium was changed every other or every third day. After the last passaging step, cells were split into a 96-well plate for the readout assay.
Primary mRECs (C576065, Cell Biologics) were cultured in Complete Mouse Endothelial Cell Medium (M1168, Cell Biologics) at 37°C in an incubator containing 5% CO₂. The medium was changed every other day until the cells were confluent for use. For the flow cytometry cell senescence assay and the ELISA experiment, mRECs were seeded in a 6-well plate at a concentration of 0.2 million cells per well. For the confocal imaging cell senescence assay, mRECs were seeded on a cell culture cover glass (NC0620709, Thermo Fisher Scientific) and laid on the bottom of a 24-well plate (3527, Corning) at 0.1 million cells per well. After 24 hours of incubation, mRECs were treated with 0.1% DMSO (control), cGAMP (20 μg/mL), STING inhibitor (2 μg/mL; inh-h151, Invivogen), or IFN-β (1000 U/mL; 12400-1, R&D Systems) for an additional 1, 2, or 4 days, respectively.

Mouse microvascular endothelial cells (Cell Biologics #C57-6024; Chicago, IL, USA; 2.5 × 10⁴ cells/well, n = 2 technical replicates per condition) were seeded in 48-well plates coated with basement membrane extract (Matrigel^® BD #354230; Franklin Lakes, NJ, USA). Cells were stimulated with either basal endothelial cell medium (CellBiologics #M1168), basal medium supplemented with 10% cardiac fibroblast secretome from the same samples used for RNA-Seq (n = 3 biological replicates), or endothelial growth medium (basal medium supplemented with VEGF, endothelial cell growth supplement, heparin, epidermal growth factor, hydrocortisone, l-glutamine, antibiotics, and FBS). Thbs1 was inhibited with a blocking antibody (10 µg/ml; Novus Biologicals #100-2059) as described previously [32 (link)]. As negative controls, endothelial cells were stimulated with the Thbs1 blocking antibody alone, an IgG1 isotype control (R&D #MAB002) alone, or basal medium diluted 1:10 with DMEM + 0.1% FBS. Images were acquired at 10X magnification following 22 h of stimulation. Angiogenesis variables were quantified using the ImageJ Angiogenesis Analyzer feature (http://image.bio.methods.free.fr/ImageJ/?Angiogenesis-Analyzer-for-ImageJ&artpage=2-6).