Lumat lb9507

The luciferase reporter vector incorporating fragments of the MYC promoter into the pGL4 vector (Promega) was utilized [16 (link)]. The CRISPR activation system (plenti-dCas9-VP64, plenti-MS2-p65-HSF1, and pE1-U6-gRNA-MS2 [28 (link)] of the individual candidate genes) or pT3.5 overexpression vectors were transfected to HEK293T cells expressing the luciferase reporter vector pRL Renilla using the Lipofectamine 3000 reagent (Invitrogen). Cells were collected 48 h after transduction. Luciferase activity was measured by following the protocol of the dual-luciferase reporter assay system (Promega) and Lumat LB9507 (Perkin Elmer). The luciferase activity values were standardized with the luciferase activity value of pE1-h-HPRT.

The fragments of the MYC promoter was integrated into a pGL4 vector (Promega). A pT3.5-CAG-M1AP was generated with pENTR221-M1AP and pT3.5-CAG-DEST (Kurata et al., 2018a ) using Gateway^® LR clonase. The luciferase reporter vectors, the pT3.5-CAG-M1AP vector, and the pRL Renilla luciferase reporter vector were co-transfected in the HEK 293T cells. The samples were harvested 48 and 72 h after the transfection. Each assay was biologically triplicated and repeated. Luciferase activities were measured using the dual-luciferase reporter assay system (Promega) and Lumat LB9507 (Perkin Elmer). RNA was harvested at the same time points and the expression of MYC mRNA was analyzed by qPCR.