Egm 2 singlequots supplement

HUVECs were seeded in 12-well plates with EBM-2 Basal medium (CC-3156, Lonza) supplemented with EGM-2 SingleQuots Supplements (CC-4176, Lonza) for 24 h. Cells were then stimulated with 5 ng/ml bFGF (Biolegend) or 5 ng/ml bFGF in combination with either 50 or 100 µm K5 for 60 min. The cells were rinsed with PBS and lysed in cell lysis buffer containing IC diluent #12 (Reagent diluent concentrate 2 DY995, R&D Systems, Minneapolis, MN, USA, in distilled water), plus 10 µg/ml aprotinin (Ref.4139, R&D Systems, Minneapolis, MN, USA) and 10 µg/ml leupeptin (Ref.1167, R&D Systems, Minneapolis, MN, USA). FGFR1 phosphorylation was measured by a sandwich ELISA (DYC5079-2, R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

Mycoplasma tested human umbilical vein endothelial cells (HUVECs; Commercially obtained from Lonza #CC‐2935) were cultured in EBM‐2 Basal Media (#00190860, #cc‐4176, Lonza, mycoplasma free) with EGM‐2 Single Quots supplements (Lonza #cc‐4176) on 0.2% gelatin (Sigma Aldrich #G1393)‐coated 96‐well plates (5,000 cells/well) or 6‐well plates (150,000 cells/well). After obtaining stable culture conditions, HUVECs were incubated with culture supernatants collected from three groups of mouse myofibroblasts: (i) untreated myofibroblasts (TurboFect), (ii) control siRNA‐treated myofibroblasts, and (iii) myofibroblasts treated with siRNA against PDGF‐Rα. For caspase activity assay, incubation with 40 μg/ml anti‐VEGF (C‐1) antibody (#sc‐7269, Santa Cruz) served as a positive control. Caspase activity was assessed after 6 h using the Caspase‐Glo 3/7 assay kit (#G8091, Promega). After 6 h of incubation, caspase activity was assessed using the Caspase‐Glo 3/7 assay kit (Promega GmbH, Germany #G8091) according to the manufacturer's instructions. Cells plated in 6‐well plate were lysed after 6‐h incubation, and lysate was processed for immunoblot analysis with cleaved caspase‐9 (Cell Signaling Technologies #9509) and eNOS (Cell Signaling Technologies #880) protein.

HEK293T (293T) cells were purchased from ATCC (Manassas, VA, USA) and maintained in Dulbecco's modified Eagle's medium (DMEM, Life Technologies, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS, Invitrogen, Carlsbad, CA, USA) and 100 unit/ml penicillin/streptomycin. HUVECs were purchased from Lonza Bioscience (C2519A, Basel, Switzerland). HUVECs were maintained in EBM-2 Basal Medium (CC-3156, Lonza Bioscience) with EGM-2 SingleQuots Supplements (CC-4176, Lonza Bioscience). All cells were cultured at 37°C in a humidified incubator with 5% CO₂.

Three different batches corresponding to three different-original donors of Human umbilical vein endothelial cells (HUVECs: CRL-1730, ATCC) were used. HUVECs were cultured in Endothelial Cell Growth Basal Medium-2 (EBM-2: CC-3156, Lonza, Bioscience) supplemented with EGM-2 SingleQuots Supplements (CC-4176, Lonza, Bioscience). All experiments were performed until the passage 10. Triple-negative breast cancer (MDA-MB-231: HTB-26™, ATCC) were used as tumor models, being cultured in Dulbecco’s Modified Eagles Medium (DMEM) (41965-039, Gibco, Life Technologies), supplemented with 10% fetal bovine serum (FBS) (S 0615, Merck), 1% Antibiotic-Antimycotic (AA; P06-07300, PAN Biotech) and 50 µg/mL Gentamicin (15750-060, Gibco, Life Technologies). Cell cultures were maintained at 37°C in a humidified environment of 5% CO₂. Cells were detached with 0.05% Trypsin-EDTA 1 × (25300-054, Invitrogen, Thermo Fisher Scientific) at 37°C for approximately 5 min and split to new plates according to the experimental procedures.
Regarding experimental conditions, cells were cultured with 15 μM hydrogen peroxide (H₂O₂; 1.07210.0250, Merck), as a ROS generator, 1.5 μM Erastin (E7781, Sigma) as a ferroptosis inducer, 100 μM Propranolol (P8688, Sigma Aldrich) and 160 and 200 μM SeChry@PURE_G4, for 6 and 16 h.

Human AoSMCs (CC-2571), human umbilical vein endothelial cells (HUVEC, CC-2517A), smooth muscle cell basal medium (SmBM-2, CC-3181), SmBM-2 plus SingleQuots of supplements (CC-3182), EBM-2 Basal Medium (CC-3156), and EGM-2 SingleQuots Supplements (CC-4176) were purchased from Lonza. Recombinant Human TGFβ1 (240-B), TNFα (210-TA), IL-1 beta (201-LB), and PDGF-BB (520-BB) were purchased from R&D Systems. Cell Titer-Glo 2.0 Assay kit was purchased from Promega (G9242). Transwell (12-mm diameter, 3.0 μm pore size) polycarbonate membrane insert was from Corning (3402). The following products were from Thermo Fisher Scientific: Total exosome isolation reagent (4478359), Total exosome RNA and protein isolation kit (4478545), scrambled microRNA control (AM4635), hsa-miR-548ai Mimic (Assay ID: MC22241), hsa-miR-548ai inhibitor (Assay ID: MH22241), Opti‐MEM I Reduced Serum Medium (31985062), Lipofectamine RNAiMAX Transfection Reagent (13778150), TaqMan MicroRNA reverse transcription kit (4366596), TaqMan Universal Master Mix II (4440043), TaqMan primers (hsa-miR548ai, Assay ID: 464169_mat; hsa-miR-544A, Assay ID: 002265; hsa-miR-4719, Assay ID: 464187_mat; hsa-miR-6886, Assay ID: 465086_mat; RNU44, Assay ID:001094).