For cloning of lentiviral overexpression constructs, first, the wild-type cDNA of REN (renin) was cloned into pLenti-C-Myc-DDK-IRES-Puro (OriGene) plasmid by digestion with Asc I and Mlu I. For the lentivirus production, 5 × 105 human embryonic kidney (HEK) 293T/17 cells were cultured in one well of a six-well plate in high-glucose Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) FBS. Twenty-four hours after seeding, the HEK293T/17 cells were transfected using Lipofectamine 2000 (11668019, Thermo Fisher Scientific) and 1.2 mg of pLenti-C-Myc-DDK-IRES-Puro-REN plasmid, 1.2 mg of psPAX2 (#12260, Addgene), and 0.8 mg of pMD2.G (#12259, Addgene) packaging plasmids. Forty-eight hours after transfection, the viral particle containing supernatant was collected, cleared by centrifugation at 500g for 10 min, and filtrated (45 mm; 09-720-005, Thermo Fisher Scientific). The viral titer was estimated using the Lenti-X GoStix Kit (#631244, Clontech). For viral transduction, Flp-In 293 T-Rex cells were infected with the virus at multiplicity of infection ~0.4 and ~1. Lentivirus-transduced cells were selected with puromycin (2 mg/ml) starting 2 days after viral infection. Expression of the C-terminal Myc-DDK–tagged REN protein was verified by Western blot using anti-MYC antibody.

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