Dual glo luciferase assay system e2940

Tcam2-cells were double transfected using the Surefect transfection reagent according to manufactures protocol (Nunclon Surface, Nunc). Cells were transfected with CIP2A-promoter construct (1802 bp upstream [38 (link)], renilla plasmid and siRNA (either scrambled or siOct4-1). Promoter construct (200 ng), renilla (10 ng) and 2 pmol of siRNA were transfected per 96 well plate. Transfections with −1802 bp, −865 bp and −1802ΔCIP2ALuc CIP2A promoter constructs were also done as described above only without siRNAs. −1802ΔCIP2ALuc construct was produced by GenScript mutagenesis service from −1802CIP2ALuc promoter construct and resulting promoter sequence was validated by DNA sequencing. After 3 days the promoter activity was measured using Promega's Dual-Glo luciferase Assay system (E2940) according to manufactures protocol. Luminescence was measured with Victor-multilabel counter 1420 (PerkinElmer).

The ALDH1L1 promoter reporter construct was generated by inserting the human ALDH1L1 promoter (including 1 kb upstream) into pLenti6-MINp-FLuc-Rluc-TKp. The pLenti6-MINp-FLuc-Rluc-TKp vector was constructed by inserting the herpes simplex virus (HSV)-thymidine kinase (TK) promoter (TKp) and Renilla luciferase (Rluc) in the opposite directions in the pLenti6-MINp-FLuc vector. The pLenti6-MINp-FLuc vector was constructed by inserting a minimal promoter (MINp) and firefly luciferase (Fluc) into pLenti6-GFP (Addgene Plasmid #35637) in place of the CMV enhancer, CMV promoter, and GFP.
Firefly and Renilla luciferase activities were measured using the Dual-Glo Luciferase Assay system E2940 (Promega, Madison, WI, USA). Briefly, after treatment with KRAS siRNA for 24 h, 75 μL Dual-Glo luciferase reagent was added to each well, and the plates were incubated for 10 min at room temperature. After measurement of firefly luminescence, Dual-Glo Stop & Glo reagent was added to the plate. After incubation at room temperature for 10 min, Renilla luminescence was measured, and the ratio of firefly to Renilla luminescence was calculated.
In silico transcription factor binding suite predictions were performed with ConSite (http://consite.genereg.net).

Wide‐type IGF‐1 (IGF‐1‐WT), mutated‐type IGF‐1 (IGF‐1‐Mut), has‐miR‐338‐3p mimics and NC mimics vectors were constructed and cotransfected in 293T cells in 96‐well plates using lipo2000 (Life, USA). At 48h after transfection, the luciferase was detected using Dual‐Glo@Luciferase Assay System E2940 (Promega, USA) according to manufacturer's protocol.