Transit lt1 transfection reagent

Cells were cultured in steroid-depleted medium for three days and seeded in 6-cm dishes (10,000 cells/dish). Twenty-four hours after seeding, cells were transfected with a plasmid encoding a luciferase reporter driven by the estrogen response element or pRL-tk-luciferase in medium containing TransIT LT-1 transfection reagent (TaKaRa). Forty-eight hours after transfection, luciferase activity was measured in triplicate using a luciferase assay system (Promega, Madison, WI, USA).

Lenti-X 293T cells were co-transfected with pDON-5 Neo plasmids encoding each TRIM5 molecule, pGP packaging plasmid (TaKaRa, Cat# 6160), and pMD2.G plasmid with TransIT-LT1 Transfection Reagent (TaKaRa, Cat# V2300) in Opti-MEM. The supernatant was filtrated 2 days after transfection. The collected retroviral vectors were used to infect CRFK cells. The cells were cultured in the presence of 500 µg/mL G-418 (Nacalai Tesque, Cat# 09380–44) for 6 days. Then, single-cell cloning was performed, and the expression of TRIM5 in each clone was evaluated by Western blotting.

The 1.47 kb DNA fragment of the Myod1 gene (−1264 to +210 from the transcriptional start site at +1) or the 1.55 kb DNA fragment of the Myog gene (−1512 to +34 from the transcriptional start site at +1) were amplified by PCR (primer sets are shown in S3 Table) from L6 genomic DNA, and they were subcloned into the luciferase reporter plasmid pGL4.16 (Promega, Madison, WI, USA) (designated pGL4.16-Myod1 Pro1474 or pGL4.16-Myog Pro1546). L6 cells were seeded on culture flasks, and cultured in growth medium for a day. Then reporter constructs were transiently transfected into L6 cells using TransIT^®-LT1 Transfection Reagent (TaKaRa Bio, Inc., Shiga, Japan). The renilla luciferase vector (pRL-SV40, Promega) was used as a transfection efficiency control. After incubation for a day, growth medium was replaced with differentiation medium and incubated for one more day under 1 G or 10^-3G conditions. Luciferase luminescence was measured using a single-sample luminometer, the Biolumat LB 9505 (BERTHOLD TECHNOLOGIES GmbH & Co. KG, Bad Wildbad, Germany) with the Dual-Luciferase Reporter Assay System (Promega). Promoter activities were calculated as the ratio of firefly to renilla luciferase readings, and the averages of at least three independent experiments were calculated. Methods were approved by a Hiroshima University Gene Recombination Experiment Safety Committee.

To obtain stably AQP8-knocked down cells, 3T3-L1 cells were transfected with a plasmid producing short hairpin RNA targeting AQP8. The plasmid was designed on pBAsi-mU6 Neo vector by TaKaRa and the target sequence was CTATCGGTCATTGAGAATA. As the negative control cells, 3T3-L1 cells were transfected with a plasmid including a nonsense sequence, TCTTAATCGCGTATAAGGC. Cells were seeded in 6-well plates two days before transfection. For each well containing the cells in about 60% confluency, 3 µg of the plasmid DNA in TransIT-LT1 Transfection reagent (TaKaRa) was added. Stably knocked down cells were selected by culturing in the presence of 3.5 mg/ml of G418, a neomycin analog (Geneticin, GIBCO) and maintained with 1.0 mg/ml of G418. For AQP8-over expressed cells, 3T3-L1 cells were transfected with a pcDNA3.1(+) vector carrying mouse AQP8 cDNA and the cells stably expressing AQP8 were selected as described above.