Endothelial cell basal medium

Umbilical cords were obtained from the maternity ward of the Kuopio University Hospital. The collection, cell extraction and following experiments were approved by the Research Ethics Committee of the Hospital District of Northern Savo, Kuopio, Finland. Informed written consent was received from all participants and the experiments were performed in accordance with the relevant guidelines and regulations. Human Umbilical Vein Endothelial Cells (HUVECs) were extracted with collagenase (0.3 mg/ml) digestion immediately after the cord collection. The cells were cultivated in Endothelial Cell Basal Medium (Lonza) with recommended supplements (EGM SingleQuot Kit Supplements & Growth Factors, Lonza). At cell extraction, Sample 0 (S0) cells were adhered for 2 h on fibronectin-gelatin coated T25 flasks, and the samples were collected after rigorous washing. S1 samples were collected at the first cell passage, and S2 and S3 on the following two passages. For microRNA sequencing, cells of four donors were collected. On one donor, the cell yield was too low for S0 sample, and therefore the number of biological replicates is three for S0, and four for S1–S3.

The HUVEC-TERT2 cell line was purchased from Evercyte GmbH (Vienna, Austria) and cultured and maintained in endothelial cell basal medium (Lonza) as described previously [49 (link), 50 (link)]. Briefly, cells were seeded into a white 384-well solid bottom plate (Nunc, ThermoFisher) at a density of 1000 cells/well in 39 μL of media using a Janus liquid handler (PerkinElmer). Serial dilutions using 1 μL of compound 1 and 2 at varying concentrations were dispensed into each well in triplicate. After 48 h incubation, 40 μL of CellTiter-Glo reagent (Promega) was added into each well. The contents were mixed for 2 min on a microplate shaker to induce cell lysis and further incubated at room temperature for 10 min to stabilize the luminescent signal. Luminescence was measured with an EnVision plate reader (PerkinElmer) and %inhibition calculations were performed using the following formula for single-point normalization: %Inhibition = (1-Raw Sample Value/Mean of DMSO Signal Reference Value) × 100. Dose response curves including EC₅₀ calculations were processed using GraphPad Prism software.

Primary antibodies were procured as follows: Ninj1 (R&D Systems INC. Minneapolis, MN, USA), p53 and GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA), p21 and tubulin (Cell Signaling Technology, Danvers, MA, USA), and CD31 (Dianova, Hamburg, Germany). Human umbilical vein endothelial cells (HUVECs; Lonza, Walkersville, MD, USA) were maintained in a gelatin-coated dish with endothelial cell basal medium (Lonza) containing endothelial cell growth supplement. The human EA.hy926 hybrid cells and human monocytic cell line THP-1 cells were obtained from the American Type Culture Collection (Manassas, VA, USA), and I-HUVEC (immortalized HUVEC) was obtained from Applied Biological Materials (Richmond, BC, Canada). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin (GIBCO Invitrogen, Grand Island, NY, USA). The cells were irradiated with a dose of 1, 2, or 5 Gy using Novalis Tx™ (Varian, Palo Alto, CA, USA) equipped with a 2.5-mm width MLC. Irradiation was delivered using a 6-MV photon beam at a dose rate of 600 monitor unit/min (Mus/min), and the average required treatment time was 32, 64, and 160 s respectively, for 1, 2, and 5 Gy.

Endothelial cells (HUVECs; Lonza Group Ltd.) were cultured in Endothelial Cell Basal medium (EBM; Lonza Group Ltd.) supplemented with an EBM-bullet kit (Lonza Group Ltd.) and 10% FBS (BioWest, Nuaillé, France) at 37 °C in a humidified 5% CO₂ atmosphere. For in vitro studies, HUVECs were seeded into six-well plates (4 × 10⁵ cells per well). All the experiments were performed after reaching 100% confluence.
For coculture experiments, PBMCs were isolated from r-axSpA patients (n = 5) and HDs (n = 5) as previously described in this section. Then, PBMCs were seeded into Transwell inserts (Corning^® Transwell^® polycarbonate membrane cell culture inserts, Sigma-Aldrich) (1 × 10⁶ cells per Transwell) in EBM, before adding them into multiple-plate wells preloaded with HUVECs for 24 h. At the end of the experiments, HUVECs were harvested for RNA and protein determinations or processed for flow cytometry.