Express five serum

Insect High Five cells (Invitrogen) were cultured at 28°C in Express Five serum-free medium (SFM, Invitrogen) containing L-glutamine (Invitrogen). The cells were cotransfected with the EGFP, EGFP-Tube, or EGFP-ΔTube plasmid and the synthesized miR-217 (miR-217-mimic) using the Cellfectin transfection reagent (Invitrogen) according to the protocol of the manufacturer. The concentrations of miR-217-mimic and the plasmid were 200 nM/well and 400 ng/well, respectively. As a control, the control miRNA was included in the cotransfection. All the miRNAs were synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China). At 48 h after cotransfection, the EGFP fluorescence of the cells was measured by a Flex Station II microplate reader (Molecular Devices, USA) at 490/510 nm of excitation/emission (Ex/Em).

Insect High Five cells (Invitrogen) were cultured in Express Five serum-free medium (Invitrogen) containing l-glutamine (Invitrogen) at 27°C. At 70% confluence, the cells were co-transfected with 2 µg of EGFP, EGFP-target gene 3′UTR or EGFP-Δtarget gene 3′UTR and 100 pM of miR-12 or miR-12-scrambled using Cellfectin transfection reagent (Invitrogen) according to the manufacturer’s instructions. The miRNAs were synthesized at Shanghai GenePharma Co., Ltd. (Shanghai, China). At 48 h after co-transfection, the fluorescence intensity of cells was assessed using a Flex Station II microplate reader (Molecular Devices, USA) at 490/510 nm excitation/emission (Ex/Em). The experiments were biologically repeated three times.