Notes: The authors identified single nucleotide mutations in gene regulatory regions that may result in transcriptional changes in the context of lung adenocarcinoma. Further, 31 of these regulatory region mutations were found in clinical lung adenocarcinoma isolates and were associated with patient outcomes. The Nano-Glo® Dual-Luciferase® Reporter Assay was used to compare expression with wild-type or mutant regulatory regions. Specifically, the relative activity of the mutant NFATC1 regulatory region was shown to be 3-fold higher compared to wild-type. (5092)

Notes: Researchers studied the relationship between Reactive Oxygen Species, cell movement and cell death using ViaFect™ Transfection Reagent for transient transfections and CytoTox-Glo™ Cytotoxicity Assay to assess cell death. (5041)

Notes: Activation of Notch isoforms via histone deacetylase inhibitors (HDACi) has been shown to suppress neuroendocrine (NE) cancers. Here, a novel HDACi called thailandepsin A (TDP-A) was characterized and shown to induce cell cycle arrest and apoptosis. The NanoBRET Target Engagement system was used to determine TDP-A binding to HDAC1. TT cells expressing an HDAC1-NanoLuc fusion were treated with TDP-A and competed HDAC tracer compound to determine in vivo binding affinity.  (5131)

Notes: ViaFect™ Transfection Reagent was used to transfect expression vectors into gall bladder cancer lines, GBC-SD and EH-GB1. Details of the transfection were not provided. (4659)

Notes: Mouse embryonic fibroblasts were transfected with expression vectors using the ViaFect™ Transfection Reagent and cultured post-transfection with zeocin to isolate stable clones. (4665)

Notes: Hs766T cells were transfected with a purchased HaloTag®-ARHGEF15 fusion expression vector in pFN21A and siRNA expression plasmids directed to the ARHGEF15 or nonsense sequence using the ViaFect™ Transfection Reagent (no details provided)., ARHGEF15 expression was visualized but the specific HaloTag® Ligand was not specified. (4667)

Notes: The ViaFect™ Transfection Reagent was used to transfect DNA into primary mouse cortical neurons 2 days after dissociation (details not provided). (4664)

Notes: The ViaFect™ Transfection Reagent was used to transfect Firefly experimental and Renilla control vectors into SW620 to judge the effects and confirm the location of a shRNA to SIRT 1 (introduced through lentiviral vectors) on the SIRT1 promoter. Reporter activity was monitored with the Dual-Luciferase® Reporter Assay System. No details of the transfection were provided. (4656)

Notes: Putative enhancer elements were cloned into the pGL4.23 [luc2/minP] and transfected into human iCMs (Cellular Dynamics) along with a Renilla luciferase control. The iCMs were plated at 2 × 10³ per well of a 96-well plate and cultured until all cells were electrically connected and beat simultaneously (~7 days post plating). Beating cells were transfected with the two reporters using the ViaFect™ Transfection Reagent at a 2:1 Viafect:DNA ratio (further detail provided in the paper). The reporter activities were measured 24 hours post-transfection with the Dual-Luciferase® Reporter Assay System. Transfections using a GFP-expressing vector provided a visual estimation of the transfection efficiency and 65-70% of the iCMs were GFP-positive 24 hrs post-transfection. (4671)

Notes: ViaFect™ Transfection Reagent was used to transfect HepG2 cells in 24 well plates with two plasmids expressing Cas9 with STAT1 or STAT3 sgRNA and the other a puromycin resistance gene expression vector. After 36 hours, puromycin was applied and following two days, diluted into 96 well plates at ~1 live cell per well. Western blotting and DNA sequencing were performed to determine gene knockout. (4658)

Notes: Mouse bone marrow macrophages (BMMs) and alveolar macrophages (AMs) were transfected with various expression vectors using ViaFect™ Transfection Reagent (details not provided). Forty-eight hours post-transfection, cells were infected with Legionella pneumophila. (4669)

Notes: To express the pank2 gene product, expression vectors were transfected into COS7, HeLa and SH-SY5Y cells using ViaFect™ Transfection Reagent (10⁵ cells per chamber of glass slides; 1µg expression vector DNA: 3µl ViaFect™ Reagent). The cDNA clones used in this study were amplified by RT-PCR using ImProm-II™ Reverse Transcriptase for cDNA synthesis and Pfu DNA Polymerase for PCR. PCR products were cloned into the pGEM®-T Easy Vector. (4663)

Notes: HEK 293T cells were transfected with various expression vectors in this study using the ViaFect™ Transfection Reagent. Transfections were performed in 12-well plates with cells at 70% confluency at the time of the transfection. ViaFect™ Reagent was mixed with DNA at 6µl/µg of DNA. The Viafect:DNA complexes were added in a 100µl volume to each well. (4660)

Notes: The ViaFect™ Transfection Reagent was tested for transfection of siRNA into “hard-to-transfect” primary human trophoblast cells. Transfection was reported to be equal to LipoFectamine 2000 and superior to DharmaFect2, Oligofectamine and Lipofectamine RNAiMAX with this cell line. Transfection efficiency was judged with a fluorescent transfection indicator. ViaFect™ Reagent and Lipofectamine 2000 achieved 62% and 57% efficiency, respectively. Data provided in supplemental materials.

(4655)

Notes: ViaFect™ Transfection Reagent was used to transfect both HTR8/Svneo and JEG-3 cells with a MSX2 expression vector.  Transfection was performed in 35mm dishes on 70% confluent cells (lipid:DNA ratios and amount of plasmid were not reported).  The authors also report transfection of both cell lines with 100nM siRNA to MSX2 or a control siRNA with >50% efficiency as determined by fluorophore-labeled siRNA molecules. (4654)

Notes: SF2 rat dental epithelial cells were transfected with expression vectors using the ViaFect™ Transfection Reagent. Prior to transfections, cells were seeded at a density of 1 × 10⁴ cell/well in a 6-well plate. (4666)

Notes: Primary rat aortic smooth muscle cells (SMCs) were transfected with two expression plasmids using the ViaFect™ Transfection Reagent (no further details provided). (4670)

Notes: A luciferase-based mammalian two-hybrid system was assembled in HeLa and D645 cells using the ViaFect™ Transfection Reagent (1µg DNA/well; 12-well plates). The results of the experiments were read with the aid of the Dual-Luciferase® Reporter Assay System. (4662)

Notes: HEK 293T cells were transfected with a reporter plasmid (assembled in the pGL3 Basic Vector) with a consensus BCL6 binding site and a BCL6 expression plasmid using the ViaFect™ Transfection Reagent (about 2µg of DNA per well in 6-well plates at 50-60% confluency). Promoter activation was monitored with a luciferase assay control with a β-galactosidase control. Western blotting of transfected cells used the Anti-Rabbit IgG (Fc), Alkaline Phosphatase Conjugate and the Western Blue® Stabilized Substrate for Alkaline Phosphatase. (4657)

Notes: A Twist1 expression vector was transfected into ~70% confluent HSG cells, a human submandibular gland cell line, using ViaFect™ Transfection Reagent. The cells were put under G418 selective pressure 48 hours later for selection of stable clones. (4661)

Notes: CHO cells expressing the human MOR were stably transfected with the GloSensor™ -22F cAMP Vector using the ViaFect™ Transfection Reagent. Inhibition of the forskolin-stimulated intracellular cAMP accumulation in the cells through the addition of the GloSensor™ cAMP Reagent (4% vol/vol). Assays were performed in 384-well plates with 5,000 cells/well in 30µl. (4668)

Notes: In order to investigate the interaction of the Cullin-RING E3 ubiquitin ligase with Elongin A due to toxic damage to DNA, the authors chose a FRET assay consisting of mCherry-labeled Cullin-RING3 and HaloTag-labeled Elongin A (clone obtained from Promega as pFN21-TCEB3 then subcloned into another expression vector). Interaction of the two proteins was examined in UV irradiated HeLa and U2OS cells. As a control, a HaloTag Protein expression vector alone was used. Intracellular HaloTag-labeled protein visualization was accomplished with the HaloTag® R110Direct™ Ligand. Both cell lines were transfected at 50-60% confluency with 700ng of plasmid in glass bottom 35mm dishes. FuGENE® HD Reagent was used for HeLa cells and ViaFect™ Reagent was used for U2OS cells. (4674)

Notes: The interaction of NUP107 and NUP133 were investigated by several means. First, in vitro expressed, biotinylated NUP107 was mixed with FLAG-tagged NUP133 and pulled down with Streptavidin MagneSphere® Particles and blotted for reaction with Anti-FLAG antibodies. HeLa cells were transfected with a GFP-NUP107 fusion vector using ViaFect™ Transfection Reagent (no details provided) and immunoprecipitated then blotted to identify NUP133 in the pull down. Subcellular localization of GFP-NUP107 was examined as well. RT-PCR analysis of nup107 splicing variants in zebrafish relied on M-MLV Reverse Transcriptase for cDNA synthesis. (4675)

Notes: Differentiated L6 myocytes were seeded at 1 × 10⁵ cells/well in a 24-well plate containing a 4:1 ViaFect™ Transfection Reagent: DNA ratio containing 1.025µg of DNA (reverse transfection). Cells were transfected with 500ng of a pGL4.12 [luc2CP]-based Ucp3 promoter construct, 25ng pGL4.74 [hRluc/TK] vector and 500ng of an SV-driven SREBP-1 expression vector. Luciferase activities were determined with the Dual-Luciferase® Reporter Assay System. The first intron was found to contain the cis-acting elements responsible for Ucp3 repression by SREBP-1. (4673)

Notes: U2OS and H1299 cells were transfected with expression vectors and analyzed for either protein levels or mRNA levels. Cells for mRNA level detection were transfected with the ViaFect™ Transfection Reagent (details not provided). Total RNA from siRNA treated cells was isolated and used in semi-quantitative RT-PCR with the PCR portion provided by the GoTaq® Hot Start Green Master Mix. The proteins ARID3A and ARID3B were expressed in with the TNT® Coupled Reticulocyte Lysate System for use in electrophoretic mobility shift assays. (4677)