Hek293t

Stable expression of mCherry protein in HTR8/SVneo cells and GFP protein in HUVECs was induced via lentiviral transduction. The lentiviral particles were generated in HEK293T (Cat#: CRL-3216, ATCC) Gaithersburg, MD, USA) cells with a second-generation packaging system and pLVX-mCherry-C1 vector (Takara Bio, California, USA). In brief, HEK293T cells were seeded into a 100 mm cell culture dish, and the plasmids of psPAX2 (3 μg), PMD2G (3 μg) and pLVX-mCherry or -eGFP (2 μg) were transfected into HEK293T cells using Fugene 6 (Cat#: E2692, Promega, Madison, WI, USA) in a 500 μl transduction volume at 60 % confluency. After overnight incubation, the transfection medium was replaced with fresh growth medium (DMEM/F12 supplemented with 10 % FBS and 10 mM HEPES) and cells cultured for 72 h. Thereafter, the supernatant was collected, and viral particles concentrated with Lenti-X concentrator (Cat#: 631231, Takara, Mountain View, CA, USA). HTR8/SVneo cells and HUVEC cells were then transduced with the mCherry- and eGFP- viral particles, respectively and placed in growth medium supplemented with puromycin (1 μg/ml, Cat#: P9620, Sigma, St. Louis, MO, USA). After 7 days of selection, cells with stable expression of mCherry and eGFP were harvested and frozen until further use.

HEK293T and HK2 cells were purchased from ATCC. Mouse anti-Prominin-1 MicroBeads (130-092-333, Miltenyi Biotec) were used to isolate PTCs from Cre-ERT2 ^+/− APE2 ^LSL/+ mice, Cre-ERT2 ^+/− mice, C57B6 mice, and APE2^−/− mice. The primary PTCs were cultured in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. HEK293T and HK2 cells were routinely genotyped using the Human Cell Line Genotyping System (Promega) and were used within their 6 times passage. Mouse PTCs were genotyped using the primer for transgenic mice and were used within their 3 times passage. All cell lines were used mycoplasma tested negative.