Fusion hd reagent

HK-2 cells were then transfected with 2 μg of empty vector (Mock) or PGC-1α DNA using 6 μl of Fusion HD reagent (Promega, Madison, WI, USA) in antibiotic-free DMEM-F12. Beginning 1 day after transfection, transfectants were selected in DMEM-F12 containing 200 μg/ml zeocin, which was refreshed every 3 days for 2 weeks. Colonies surviving in the selection medium were collected and sequentially plated in 48, 12, 6-well plates, and then 60 and 100 mm dishes. Cells stably overexpressing human PGC-1α were identified by immunoblotting with anti c-myc and anti β-actin antibodies or by PCR analysis using zeocin primers 5′-ATGGCCAAGTTGACCAGTGCCGTT-3′ (forward) and 5′-GTCCTGGTCCTCGGCCACGAAGTG-3′ (reverse)^{57 (link)}. A loading control was analyzed by using GAPDH primers 5′-ACCACAGTCCATGCCATCAC-3′ (forward) and 5′-TCCACCACCCTGTTGCTGT-3′ (reverse).

HK-2 cells were transfected with 2 μg of empty vector (Mock) or RON DNA using 6 μL of Fusion HD reagent (Promega, Madison, WI, USA) in antibiotic-free DMEM-F12. Beginning at 1 day after transfection, transfectants were selected in DMEM-F12 containing 200 μg/mL zeocin, which was refreshed every 3 days for 2 weeks. Colonies surviving in the selection medium were collected and sequentially plated in 48-, 12-, and 6-well plates, and then 60- and 100-mm dishes. Cells stably overexpressing human RON were identified by immunoblotting with anti c-myc and anti β-actin antibodies or by PCR analysis using zeocin primers 5′-ATGGCCAAGTTGACCAGTGCCGTT-3′ (forward) and 5′-GTCCTGGTCCTCGGCCACGAAGTG-3′ (reverse) [49 (link)]. A loading control was analyzed by using GAPDH primers 5′-ACCACAGTCCATGCCATCAC-3′ (forward) and 5′-TCCACCACCCTGTTGCTGT-3′ (reverse).