Murine TM3 cells (Cat# CRL-1714; American Type Culture Collection, Manassas, VA, USA) were cultured at 37 °C with 5 % CO2 in Dulbecco's modified Eagle's medium/F-12 (Cat# LM002-04; WelGENE Inc., Daegu, Korea), with 10 % fetal bovine serum and 1 % penicillin-streptomycin solution added. For knocking down Tas1r3, 1 × 105 TM3 cells per well were seeded in a 6-well culture plate approximately 24 h before transfection. Upon reaching 60 % confluency, each well was transfected with Tas1r3 siRNA (20 nM; Bioneer, Daejeon, South Korea) and Lipofectamine™ RNAiMAX® complexes (Invitrogen, Thermo Fisher Scientific, USA) according to the manufacturer's instructions. Following 48 h incubation, all media were collected, and the cells were harvested for subsequent biochemical analysis. The siRNAs' target sequences utilized for the knockdown are detailed in Table S2 .
Tas1r3 sirna
Manufactured by Bioneer
Tas1r3 siRNA is a small interfering RNA (siRNA) molecule designed to target and silence the Tas1r3 gene. Tas1r3 encodes the taste receptor type 1 member 3 protein, which is involved in the perception of sweet and umami tastes. The Tas1r3 siRNA can be used as a research tool to study the role of the Tas1r3 gene in various biological processes.
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2 protocols using tas1r3 sirna
1
Silencing Tas1r3 in Murine TM3 Cells
2
Knockdown of Tas1r3 in mHypoA-2/12 cells
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For the knockdown of Tas1r3, 3x104 mHypoA-2/12 cells per well were seeded in a 6-well culture plate approximately 24 hr prior to transfection. After the cells had grown to 50–60% confluency, 40nM of Tas1r3 siRNA (Bioneer, Daejeon, Korea) and negative control siRNA (Bioneer) complexed with Lipofectamine RNAiMAX® (Invitrogen, ThermoFisher Scientific, USA) were added to each well according to the manufacturer’s instructions and incubated for 48 hr at 37 °C. On the day of the experiments, the transfection medium was removed, and the supernatants were replaced with no glucose medium (Cat # LM001-06, WelGENE) or medium containing 10mM glucose, 10mM fructose and 10μM palmitate. The cells were then incubated for 0, 12, and 24 hr at 37 °C. Following incubation, cell viability at each time point was measured using CountessTM 3 Automated Cell Counters (Invitrogen). Then, the cells were washed with phosphate buffered saline, and RNA was isolated for qPCR. The target sequences of the siRNA are listed in Additional file 2 : Table S2.
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