Pgl4.20 luc2 puro vector

The plasmids pA3F-EBNA3C, pA3F-EBNA3C 1–365, pA3F-EBNA3C 366–620 and pA3F-EBNA3C 621–992 encoding full-length Flag-tagged EBNA3C and truncated mutations of EBNA3C have been described previously [65 (link)]. The full-length RASSF1A was amplified from total RNA by reverse transcription PCR via using primers 5’-CGCGGATCCATGTC-GGGGGAGCCTGAG-3’ and 5’-CCGCTCGAGACTCCCCAGAGTCATTTTCCTTCAGG-3’. The PCR products were cloned into pA3M and pEGFP-N1, respectively. The pA3M-RASSF1A cDNA was used as the template for PCR amplification to generate RASSF1A mutants. The PCR products were cloned into pA3M. pGL4.2-R1A-promoter used to express RASSF1A promoter was amplified from genomic DNA by primers: 5’-GAAGATCTAGCCCAGGGTGACAGAGCCA-AATG-3’ and 5’-CCAAGCTTGGCCCGGTTGGGCCCGTGCTTC-3’. PCR products were inserted into pGL4.20[luc2/Puro] vector (Promega Corporation, Madison, USA) by Bgl II and Hind III.

The 5’ flanking region of PADI3 gene was amplified by PCR using the Q5 High-Fidelity DNA polymerase (New England Biolabs, Ipswich, USA), the human genomic DNA from HFFs as a template, and the PAD3 primers containing Xho I and Hind III restriction enzymes sites (see primers sequences listed in S1 Table). The resulting amplification products were digested with Xho I and Hind III (Thermo Fisher Scientific, Waltham, USA) and cloned into the pGL4.20[luc2/Puro] vector (Promega, Madison, USA), which encodes the luciferase reporter gene luc2 (Photinus pyralis) with no other regulatory elements. The resulting pGL4.20-PADI3prom construct was purified using the PureYield Plasmid Miniprep System (Promega, Madison, USA) and verified by restriction mapping and complete sequencing. The resulting chromatograms were analyzed using Chromas software 2.6.6 (Technolysium Ltd.).

Putative region of SOS1 (−1000 bp to +200 bp of transcription start site) was cloned from the genomic DNA of K562 cells using the following primers; F 5′-CTCCCTGCAGAAATCCCGAG-3′ and R 5′-TCTTGGGACAAAACAAAACACCT-3′, and then subcloned into pGL4.20 [luc2/Puro] vector (Promega). Both pGL4.20 SOS1 promoter vector and pRL-CMV control vector (TOYOBO B-Net Co., LTD.) were co-transfected into HEK293T cells that are stably-expressing shRNA of sh_Luc or expression vector of RUNX1. Promoter activities were measured using PicaGene Dual Sea Pansy Luminescence Kit (TOYOBO B-Net Co., LTD.) and detected by ARVO X5 (Perkin Elmer) according to the manufacturer’s instructions.

Promoter regions of BIRC5 and PIF1 including the RUNX1-binding consensus site (5’-TGTGGT-3’) and transcription start site were cloned from the genomic DNA of A172 cells using the following primers; F 5′-CAGAAAATCTGGGTGAAGGGTATATGAG-3′ and R 5′-GATGCGGTGGTCCTTGAGAAAG-3′ for BIRC5, F 5′-AAGAACCTGGACAACTTTCAGTCATCA-3′ and R 5′-CCGACACTCAGATGAACAAGCAGAT-3′ for PIF1, then subcloned into pGL4.20 [luc2/Puro] vector (Promega, Madison, WI). These pGL4.20 vectors and pRL-CMV control vector (TOYOBO) were co-transfected into HEK293T cells transduced CSIV-TRE-Ubc-KT lentivirus expression vector (RIKEN BRC) tet-inducibly expressing RUNX1 cDNA using ViaFect Transfection Reagent (Cat#E4981; Promega) 48 h after RUNX1 expression. Promoter activities were measured 16 h after transfection using a Dual-Glo Luciferase Assay System (Promega) and detected by Spark multimode microplate reader (Tecan, Männedorf, Switzerland) according to the instructions from the manufacturer.

Five regulatory element deletion fragments were amplified using primers (Additional file 3: Table S3). The purified PCR products were digested with KpnI and XhoI and ligated into the pGL4.20[luc2Puro] vector (Promega, WI, USA). For the binding site mutation assay, synthesized DNA sequences containing mutated binding sites were cloned into the pGL4.20[luc2Puro] vector.
Recombinant plasmids and internal control vector PRL-TK Renilla vector were transfected into Jurkat cells using Lipofectamine 2000 reagent (Invitrogen, USA) following the instructions. Cells were harvested at 48 h post-transfection and assayed for luciferase activity using the Dual-Luciferase Reporter Assay System (Promega, USA).