Sirna duplex oligonucleotides

Cells were transfected with plasmids or siRNA oligonucleotides using Lipofectamine2000 (Invitrogen). For Luciferase reporter assays, the pRL-TK renilla luciferase was served as a normalizing control. One day after transfection, cells were treated with MG132 for an additional 2 h, and cell lysates were subjected to dual luciferase assays (Promega). For RNA interference, siRNA duplex oligonucleotides or scramble oligonucleotides were synthesized (Invitrogen). In total, 100 pmol siRNA was added in one well from a six-wells plate. The siRNA sequences are provided in Supplementary Table 3.

The small interfering RNA (siRNA) duplex oligonucleotides targeting human ChREBP were selected and synthesized by Invitrogen (StealthTM RNAi, Carlsbad, CA, USA). The control siRNA oligonucleotides were purchased from Genolution (Seoul, Republic of Korea). The siRNA sequences were as follows: siChREBP forward 5′-CCAAGUGGAAGAAUUUCAAAGGCCU-3′, reverse 5′-AGGCCUUUGAAAUUCUUCCACUUGG-3′; siC forward 5′-CCUCGUGCCGUUCCAUCAGGUAGUU-3′; reverse 5′-CUACCUGAUGGAACGGCACGAGGUU-3′. For gene silencing or overexpression, Caco-2BBE cells were transfected with siRNAs or plasmids containing human ChREBP, Mlx, and β-galactosidase. The proximal region of Glut5 (–2165/+0) was amplified from mouse genomic DNA using PCR and inserted into the pGL4 basic vector (mGlut5-2165) [8]. All transfections were performed using reverse transfection. RNAiMAX (Thermo Fisher Carlsbad, CA, USA) and X-tremeGENE HP DNA transfection (Roche Diagnostics, Indianapolis, IN, USA) reagents were used for gene silencing and overexpression, respectively. Following 24 h of transfection, fresh DMEM containing 10% fetal bovine serum with 1% non-essential amino acids was added into individual wells of 24-well plates, followed by Dex treatment 24 h after media addition.

The full-length Epas1 plasmid was purchased from Youbio (Hunan, China). The pCMV-FLAG-FLCN plasmid was constructed as previously reported, using the following primer set. The above constructs were confirmed via DNA sequencing. The cells were seeded in a six-well plate, cultured to 80–90% confluence, and then transfected with Epas1 and pCMV-FLAG-FLCN plasmids, respectively, by using FuGENE HD Transfection Reagent (Promega Corporation, Madison, WI, USA).
The non-specific control siRNA and siRNAs for FLCN and EPAS1 were purchased from GenePharma (Shanghai, China). The cells were transfected with siRNA Duplex oligonucleotides using Lipofectamine 2000 (Invitrogen), according to the transfection method provided by the manufacturer. The siRNAs are listed in Table 1.