Egmtm 2 bulletkittm

A murine mesangial cell line, Mes13, was obtained from American Type Culture Collection (ATCC, Manassas, VA). Mes13 cells were cultured with Dulbecco’s Modified Eagle Medium (DMEM) containing high glucose/Nutrient Mixture F-12 Ham supplemented with 10% fetal bovine serum (FBS). Human umbilical vein endothelial cells (HUVEC) were cultured with EGM^TM-2 BulletKit^TM (Lonza, Basel, Switzerland). An adherent macrophage cell line, Raw 264.7, was obtained from ATCC and were cultured with DMEM containing high glucose concentration with 10% FBS at 37 °C under 5% CO₂. For the analysis for M1/M2 macrophage subtype transition by CTGF or TNF-α, RAW264.7 cells were stimulated with vehicle or 1000 ng/ml CTGF (PROSPEC, East Brunswick, NJ) in DMEM without FBS, and then harvested 3 h after stimulation. Stimulation with 10 ng/ml recombinant mouse TNF-α (R&D systems, Minneapolis, MA) was performed at the same protocol, and collected at 3 h. To examine mRNA expression of Ccl2 in mesangial cells, Mes 13 cell were stimulated with 1 ng/ml TGF-β1 and/or 500 ng/ml CTGF, and then harvested 3 h after stimulation.

The human bladder carcinoma cell lines, 5637 and T24, were purchased from the American Type Culture Collection (Manassas, VA, USA) and maintained in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a 5% CO₂ humidified incubator. The 5637 cells were referred to as MGH-U1 cells. Additionally, primary human umbilical vein endothelial cells (HUVECs) were obtained from Lonza (Walkersville, MD, USA). Cells were grown on plates coated with 0.1% gelatin (Sigma, San Diego, CA, USA) in endothelial basic medium (EBM) and cultured in endothelial growth medium-2 (EGM^TM 2) Bulletkit^TM (Lonza) at 37 °C in a 5% CO₂ humidified incubator. All experiments were performed between passages 2 and 5.

Human umbilical vein ECs (Pooled HUVECs, Lonza) were grown in the EC growth medium supplemented with growth factors, 5% fetal bovine serum (FBS), and antibiotics (EGM^TM‐2 Bulletkit^TM; Lonza) at 37^oC and 5% CO₂. Passage 4–6 HUVECs were used for the experiments. Cells were (60%–70%) confluent and were starved over‐night in MCDB‐131 basal medium with 1% FBS, followed by treatment with pravastatin sodium salt hydrate (10 µM; Sigma) (Abe et al., 2006; Panczel et al., 2019) in the same MCDB‐131 basal medium with 1% FBS for 24 hr. Controls were treated with vehicle phosphate‐buffered saline. To validate the expression level of top up‐ or downregulated lncRNAs and mRNAs, HUVECs were cultured and treated as previously described.