Thermo scientific vanquish system

The wild type (WT) or mutant (Mut) TET2 cDNA fragments of the target binding site predicted by circYTHDC2 were directly synthesized by Songon Biotech Corporation (Shanghai, China) and inserted into the luciferase report gene pGL3 (Promega) to construct a recombinant vector. A7R5 cells were co-transfected with circYTHDC2 siRNA (50 pmol) and wild-type or mutant TET2 (50 pmol) in a 96-well culture plate (1 × 10⁴ cells per well) using Lipofectamine 3000 according to the manufacturer's instructions. After 48 h of transfection, the luciferase activity was detected by a Thermo Scientific Vanquish system (Thermo Scientific, USA).

The Dual-Luciferase reporter gene assay, based on the methods described by Wu et al. (2021) (link) with modifications, was conducted. A recombinant vector, synthesized by Songon Biotech Corporation (Shanghai, China), containing wild-type (WT) or mutant (Mut) Caspase 3 promoter fragments corresponding to the predicted target binding site of circDNTTIP2, was used. L02 cells were transfected in a 24-well culture plate with circDNTTIP2 (800 ng) or circDNTTIP2 siRNA (20 pmol), along with either the wild-type or mutant Caspase 3 promoter (20 pmol), using Lipofectamine 2000 following the manufacturer’s instructions. After 48 h of transfection, cell harvesting was performed, and luciferase activity was measured using the Thermo Scientific Vanquish system (Thermo Fisher Scientific, Waltham, MA, USA).

The wild type (WT) or mutant (Mut) Caspase 1 cDNA fragments of the target binding site predicted by circCUX1 were inserted into the luciferase report gene pGL3 (Promega) to construct a recombinant vector. Fadu cells were co-transfected with circCUX1 (800 ng) or circCUX1 siRNA (20 pmol) and WT or Mut Caspase 1 (20 pmol) in a 24-well culture plate (1 × 10⁵ cells per well). The transfection process was carried out using Lipofectamine 2000 system. After 48 h of transfection, the cells were collected, and the luciferase activity was detected by a Thermo Scientific Vanquish system (Thermo Scientific, USA).