Pk 15

SLC38A9 and SLC36A1 overexpression constructs were amplified from mouse cDNA and cloned into pcDNA3.1. Small interfering RNA (siRNA) targeting SLC38A9 or SLC36A1 was obtained from GenePharma (Shanghai, China). For mouse, the sequences of SLC38A9 siRNA were 5′-GAUGGGAACAUCUAUACUATT-3′ and 5′-UAGUAUAGAUGUUCCCAUCTT-3′, and the sequences of SLC36A1 siRNA were 5′-GUGUGCUCAUCACUUAUGUTT-3′ and 5′-ACAUAAGUGAUGAGCACACTT-3′. For pig, the sequences of SLC38A9 siRNA were 5′-GCAGUGACAUCCUGUCCUUTT-3′ and 5′-AAGGACAGGAUGUCACUGCTT-3′, and the sequences of SLC36A1 siRNA were 5′-CCGAGAUCAUCAUUCCUUUTT-3′ and 5′-AAAGGAAUGAUGAUCUCGGTT-3′. For the SLC38A9 and SLC36A1 overexpression experiments, C2C12 cells were transfected with a pcDNA3.1 expression plasmid expressing SLC38A9 or SLC36A1, or an empty pcDNA3.1 plasmid using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), and they were harvested 48 h later. In the SLC38A9 and SLC36A1 knockdown studies, C2C12, 3T3-L1, BHK-21, PK-15, and ST cells were either transfected with control siRNA (NC) or SLC38A9 and SLC36A1 siRNA using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), and they were harvested 48 h later. The 3T3-L1 and BHK-21 cell lines were purchased from the Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). PK-15 and ST cell lines were purchased from the China Center for Type Culture Collection (Wuhan, China).

The porcine kidney cells (PK15) and the human kidney cells (293 T) were obtain from China Center for Type Culture Collection (CCTCC) and cultured using DMEM medium with high glucose (Hyclone, USA) supplemented with 10% FBS (PAN, Germany) in a humidified atmosphere of 5% CO₂ at 37 °C.