Prl tk renilla vector

A549 cells were cotransfected with p53-Luc plasmid (Agilent Technologies, Santa Clara, CA, USA) and pRL-TK Renilla vector (Promega, Madison, WI, USA) using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA). After 24 h, the transfected cells were treated with passive lysis buffer at room temperature for 10 min. Cell lysates were then transferred to a plate, and luciferase assay reagent and stop reagent were added in sequence. Next, luciferase activity was measured using a Luciferase Assay System (Promega, Madison, WI, USA) according to the manufacturer’s instructions, as previously described^{31 (link)}.

MM001 and MM047 were seeded in 24-well plates and transfected with 400 ng of pGL4.23-enhancer vector + 40 ng of pRL-TK Renilla vector (Promega) with Lipofectamine 2000 (Thermo Fisher Scientific). As positive controls, the previously published enhancers MLANA_5-I, IRF4_4-I and TYR_−9-D or ABCC3_11-I and GPR39_23-I were used for MM001 and MM047, respectively^{77 (link)}. One day after transfection, luciferase activity was measured through the Dual-Luciferase Reporter Assay System (Promega) by following the manufacturer’s protocol. Briefly, cells were lysed with 100 µl of passive lysis buffer for 15 min at 500 rpm. A total 20 µl of the lysate was transferred in duplicate in a well of an OptiPlate-96 HB (PerkinElmer) and 100 µl of luciferase assay reagent II was added in each well. Luciferase-generated luminescence was measured on a Victor X luminometer (PerkinElmer). A total of 100 µl of the Stop & Glo Reagent was added to each well and the luminescence was measured again to record Renilla activity. Luciferase activity was estimated by calculating the ratio luciferase/Renilla; this value was normalized by the ratio calculated on blank wells containing only reagents. Three biological replicates were done per condition for MM001 and two biological replicates for MM047.

The cDNA encoding N1ICD was subcloned into the expression vector pcDNA3.1(+) between the BamHI and XhoI sites, and subcloning was confirmed with sequencing by Shanghai Genechem Co., Ltd. The pGL3 reporter construct plasmid (−2000/+100) consisted of a 2100-bp genomic DNA fragment of the Slug promoter (Promega, Madison, WI, USA). The pGL3-Slug promoter plasmid or its negative control pGL3-basic plasmid carrying the firefly luciferase reporter were co-transfected with an internal control, pRL-TK Renilla vector (Promega), by using Lipofectamine 2000 (Invitrogen). In addition, cells were respectively transfected with 600 ng of N1ICD overexpression plasmid pcDNA3.1(+) or its negative control pcDNA3.1. Cell lysates were harvested 48 h after transfection. The firefly and renilla activities were measured by the Dual-Luciferase Reporter Assay System (Promega). Firefly luciferase activity was normalized to the Renilla luciferase activity. Each transfection was repeated three times.

The 3′-UTR of PTEN was amplified by PCR using genomic DNA and cloned into the NheI and XhoI sites in the pGL3 vector (Promega). The mutant 3′-UTR of PTEN was also generated by PCR and cloned into the NheI and XhoI sites in the pGL3 vector (Promega). RD cells or HEK293 cells were seeded in 96 well plates and transiently transfected with luciferase reporter plasmids (wt-PTEN-UTR-pGL3 or mt-PTEN-UTR-pGL3), the control plasmid pRL-TK-Renilla vector (Promega), and hsa-miR-494-3p mimics or negative control. After 48 h, the cells were collected and the luciferase activities were measured using the dual-luciferase reporter assay system, according to the manufacturer's instructions (Promega).

The SP1, SP3 and SUB1 ORFs were amplified from cDNA and inserted into the EcoRI and SacII restriction sites of the pEGFP-N1 plasmid (Clontech) to create SP1-EGFP, SP3-EGFP and SUB1-EGFP plasmids. The DEAF1-EGFP-N1 plasmid was kindly donated by CG Fathman (Yip et al. 2009 (link)). The GDF5 region spanning −93 to +304 relative to the TSS and encompassing the 5ʹUTR was inserted into the MluI and BglII sites of the pGL3-enhancer plasmid (Promega) to create rs143383 C and T allele 5ʹUTR-pGL3 enhancer plasmids. Primer sequences are listed in Table 1. 5ʹUTR-pGL3 plasmids were in vitro methylated as above. SW1353 cells were seeded as described (Reynard et al. 2011 (link)) and co-transfected with 500 ng of SP1-EGFP or SP3-EGFP plasmid, 500 ng of rs143383 C allele or T allele 5ʹUTR-pGL3 enhancer plasmid and 30 ng of the control pRL-TK Renilla vector (Promega, Southampton, UK) using ExGen 500 in vitro transfection reagent. Empty EGFP-N1 and pGL3 enhancer plasmids were used as a control. Five wells were transfected per condition and four independent experiments were performed. After 24 h, luciferase and renilla readings were assayed as above, normalised to the EGFP-N1 and pGL3 control plasmids.

The cDNA encoding HOXA7 was subcloned into the expression vector pBabe and subcloning was confirmed with sequencing by Shanghai Genechem Co., Ltd. The pGL3 reporter construct plasmid (−3000/+100) consisted of a 3100-bp genomic DNA fragment of the Snail promoter (Promega, Madison, WI, USA). The pGL3-Snail promoter plasmid or its negative control pGL3-basic plasmid carrying the firefly luciferase reporter were co-transfected with an internal control, pRL-TK Renilla vector (Promega), by using Lipofectamine 2000 (Invitrogen). In addition, cells were respectively transfected with 600 ng of HOXA7 overexpression plasmid pBabe or its negative control pbabe-vector. Cell lysates were harvested 48 h after transfection. The firefly and renilla activities were measured by the Dual-Luciferase Reporter Assay System (Promega). Firefly luciferase activity was normalized to the Renilla luciferase activity. Each transfection was repeated three times.