Fbs dmem f12

Mouse primary proximal tubular epithelial cells (PPTCs) were extracted from renal tissues of 4-6 mice by dissecting renal cortex as well as inner and outer medulla according to the previous literature [15 (link)]. Then, the cortex and outer medulla were digested with collagenase IV, subjected to serial sieving by cell strainer (40  μ m, BD Falcon), rinsed with Hanks' balanced salt solution (Thermo Scientific™), layered with Percoll® (Sigma-Aldrich), and cultured with 10% FBS DMEM/F12 (Gibco™) supplemented with insulin-transferrin-sodium selenite media supplement (Sigma-Aldrich), 1 mM hydrocortisone (MedChemExpress®), 50 mM vitamin C (MedChemExpress®), and 100 U/ml penicillin-0.1 mg/ml streptomycin (Gibco™). Cells at a ratio of 2 × 10⁵ viable cells/well in 6-well culture plates were used for subsequent experiments.

Gene rescue in c-Kit deficient (Kit^W∕W−v) SMC was performed using a lentiviral vector (pRVPG24). This rescue vector was constructed by inserting a blunted BsrBI-NotI digested DNA fragment (3.6 Kb), containing the coding region of the mouse Kit cDNA under the murine phosphoglycerate kinase (PGK) promoter, into the blunted EcoRV-ClaI digested pLenti CMV PuroDest vector (Addgene Inc., Cambridge, MA, USA). Third generation lentiviral stocks were produced in HEK-293 cells co-transfected with the lentiviral vector and the packaging and envelope plasmids psPAX2 and pMD2.G (Addgene Inc.). Transfections were done with the jetPRIME transfection kit (Polyplus, New York, NY). Infected cells (100 MOI) were selected in DMEM-F12-FBS (5:3:2; Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 100 I.U./ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, 0.075% sodium bicarbonate, and 10 µg/ml puromycin (Sigma, St Louis, MO).
Knockdown of TAK1 or NLK in c-Kit wild type (Kit^+∕+) SMC was performed using pooled lentiviral particles carrying different target siRNAs (Table S1; Applied Biological Materials, Richmond, Canada). An anti-GFP siRNA was used as control. Transduced cells were puromycin-selected as described above. All gene modifications were confirmed by analytical flow cytometry or Western blot (WB).

Primary aortic SMC were isolated from c-Kit deficient (Kit^W∕W−v) mice and control littermate mice (Kit^+∕+) (Stock #100410, The Jackson Laboratories, Bar Harbor, ME, USA) (Bernstein et al., 1990 (link)) using the explant technique (Metz, Patterson & Wilson, 2012 (link)) with minor modifications. Briefly, mouse aortas were digested with collagenase type II (5 mg/mL, Worthington, Lakewood, NJ, USA) at 37 °C for 1 h, after which they were transferred to 10% FBS and cut with a scalpel into small pieces. Individual SMC migrated out of the explants within 1 week of culture. Cells were maintained in DMEM-F12-FBS (5:3:2; Thermo Fisher Scientific, Waltham, MA) supplemented with 100 I.U./ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, and 0.075% sodium bicarbonate (Metz, Patterson & Wilson, 2012 (link)). Primary cells were maintained at ∼90% confluency and used within three passages to avoid fibroblast-like phenotypic switching. All animal procedures were performed according to the National Institutes of Health guidelines (Guide for the Care and Use of Laboratory Animals) and approved by the University of Miami Miller School of Medicine Institutional Animal Care and Use Committee (protocol 15-114).