Notes: Cyclin G associated kinase (GAK) is a central regulator of viral and bacterial host cell entry. The synthesis and characterization of selective and potent GAK inhibitors is described. Inhibitors were assessed for ability to compete for GAK binding using the NanoBRET Target Engagement system. Briefly, a BRET signal was observed with the NanoLuc-GAK fusion in the presence of a red-shifted dye specific to the GAK ATP binding site. Upon treatment with GAK inhibitors, the dye was competed away, and a loss of BRET signal was observed. (5130)

Notes: The NanoBiT® PPI MCS Starter System was used to build assays to study the interaction dynamics of RSK1 with ERK2 and MAGl-1 in live cells upon EGF treatment. Various structure-based mutations were studied by transiently transfecting cells with constructions using FuGENE® HD Reagent and assayed with Nano-Glo® Live Cell Reagent. The authors were able to determine which mutations in RSK1 affected MAGl-1 dissociation and determined that it was dependent on the presence of the PDZ-binding motif. (4969)

Notes: Determining target engagement of potential therapeutics and their target protein is commonly assessed through Thermal Shift Assays (TSA). Cell-based TSA (CETSA) now provide a more physiologically relevant information on target engagement however, these assays require use of antibodies which selectively identify the target protein and commonly have problems with reproducibility. The NanoLuc^® luciferase thermal shift assay (NaLTSA) uses NanoLuc^® luciferase activity to measure the remaining soluble fraction of the target protein making it a simplified procedure with higher reproducibility. (5055)

Notes: The NanoBiT Protein-Protein Interaction System was utilized to investigate the interaction of human A₃ adenosine receptor, a G protein-coupled receptor, and β-arrestin2. Specifically, the importance of specific C-terminal phosphorylation sites for A₃AR and β-arrestin2 coupling was determined using protein truncations. To determine the optimal fusion tags for this assay, multiple constructs were tested in the presence of an A₃AR selective agonist. Unlike previous studies in rat models, C-terminal human A₃AR truncations displayed no major impact on β-arrestin2 coupling. (5077)

Notes: NanoBiT Protein-Protein Interaction System was used to determine an interaction between Programmed death ligand 1 (PD-L1) and B7-1. PD-L1 is an encouraging target for cancer immunotherapy due to its involvement in mediating tumor immune evasion. Previous studies identified an interaction between PD-L1 and B7-1, however here the NanoBiT system was used to show binding between PD-L1 and B7-1 was specific when both are co-expressed on the same cell surface. Interestingly, similar co-expression of PD-L1 and B7-1 was found on tumor-infiltrating myeloid cells. Further, targeted PD-L1 mutants showed a significant decrease in B7-1 binding. (5072)

Notes: Vascular endothelial growth factor (VEGF) receptor interactions are observed using the NanoBRET™ Protein-Protein Interaction System. A novel method of labeling VEGF at a single N-terminal cysteine (TMR) to maintain full activity is presented. NanoLuc^®-VEGFR2 and VEGF-TMR are used in conjunction to monitor an interaction and internalization into intracellular endosomes. The effect of receptor tyrosine kinase inhibitors such as Cediranib and vandetanib on internalization in living cells is assessed. (5060)

Notes: Researchers set out to identify CBP/EP300 bromodomain inhibitors potent to both in vitro targets and targets in cellular target engagement assays. They developed a series of selective probes of CBP/EP300 bromodomains identified first by fragment screening. They next substituted and modified parts of the fragments to improve potency and selectivity.

To determine whether improvements in CBP bromodomain inhibition that were observed in their biochemical assay would translate to a cellular context, they used a bioluminescence resonance energy transfer assay, NanoBRET, where a small CBP/EP300 bromodomain inhibitor disrupted the interaction between a HaloTag-labeled histone and bromodomain conjugated to NanoLuc® Luciferase.

NanoBRET™ CBP/Histone H3.3 Interaction Assay (Cat.# N1850) and FuGENE® HD Transfection Reagent (Cat.# E2311) were used in the cell-based assay. (4717)

Notes: HaloTag® Pull-Down Assay
HEK293T (12 × 10⁶ cells) were plated and grown to 70–80% confluence (approximately 18 hours). The cells were then transfected (using FuGENE® HD Transfection Reagent [Cat.# E2311]) with either 30µg of HaloTag(HT)-HIF1A or HT-alone control vector (vectors available by custom order from Promega Custom Assay Services). Clarified lysates from both HT-HIF1A and HT-alone control cells were prepared and incubated with HaloLink™ Resin (HaloTag® Mammalian Pull-Down System [Cat.# G6500, G6504]). Proteins were digested with trypsin, and digestion was quenched with formic acid. Digested peptides were analyzed by mass spectrometry.

NanoBRET™ Assay
HCT116 and HEK293 cells (8 ×10⁵) were plated in each well of a 6-well plate and co-transfected with one of three acceptors: HT-Pontin, HT-Reptin or HT-TIP60, in combination with the HIF1A-NanoLuc(NL) donor. The following NanoBRET pairs used are available by custom order from Promega Custom Assay Services: HIF1α-NLuc + HT-TIP60, HIF1α-NLuc + HT-Pontin or HIF1α-NLuc + HT-Reptin. (4718)

Notes: The authors used bioluminescence resonance energy transfer (BRET) to test the ability of a bromodomain 9 ligand to disrupt binding to histone. HEK 293 cells were cotransfected with a histone H3.3-HaloTag® fusion vector and either NanoLuc®-BRD9 bromodomain or NanoLuc®-full-length BRD4 fusion vector. After 24 hours, the transfected cells were trypsinized, diluted in phenol red-free DMEM with or without 10nM of HaloTag® NanoBRET™ 618 Ligand and dispensed into a 96-well plate. One of two potential BRD-disrupting compounds, 7d or 11, was adding to a final concentration of 0.005–33μM, cells were incubated for 18 hours and NanoBRET™ Nano-Glo® Substrate (final concentration 10µM) was added. Fluorescence was measured and a corrected BRET ratio calculated. Cytotoxicity was assessed after the NanoBRET™ assay by incubating the cells with the CellTiter-Glo® Reagent for 30 minutes and measuring luminescence. To examine histone H3.3 localization, HEK 293 cells were transfected with the histone H3.3-HaloTag® fusion vector using FuGENE® HD Transfection Reagent. After 24 hours, cells were labeled with 5μM HaloTag® TMR ligand for 15 minutes before washing with complete medium, incubated for 30 minutes and imaged with a confocal microscope. (4568)

Notes: Total RNA was isolated from HTC-15 human colorectal carcinoma cells with the ReliaPrep™ RNA Cell Miniprep System. Isolated RNA was used in an RT-PCR method to amplify alternative splice variants of the target transcript. Once identified, a RT-qPCR experiment was designed to look for the major splice variants in normal cell lines derived from 15 human tissues (RNA isolation details not provided, assumed to be the same as HTC-15). The alternative splice variants were overexpressed in HTC-15 cells by transfection via FuGENE® HD Transfection Reagent and noted a decline in full-length expression. (4607)

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: Researchers were interested in developing a protocol to test transfection efficiency, cellular toxicity and stress response of transfection reagents. Using an EGFP DNA plasmid transfected into HeLa cells as their model system, seven different transfection reagents including FuGENE® HD Transfection Reagent were assessed using whole well imaging, Hoechst staining of nuclei and immunostaining with multiplexed readout. The transfection reagents were tested in parallel at 0.025, 0.05 and 0.1μl/well using EGFP plasmid or scrambled siRNA duplex, and compared to negative control wells (no nucleic acid). Transfection efficiency and cellular effects including morphology were assessed 24, 48 and 72 hours post transfection. Of the panel of reagents, FuGENE® HD Transfection Reagent had the highest transfection efficiency and according the authors “emerged as the most optimal reagent with no apparent side effects suitable for performing microtiter based miniaturized transfection for both chemical and RNAi screening”. (4766)

Notes: The authors transiently transfected 2 × 10⁵ HeLa cells grown on coverslips in a 6-well plate using the FuGene® HD Transfection Reagent, 2.5µg of DNA and a reagent:DNA ratio of 8:2.5. (4433)

Notes: The HaloTag^® protein tag was used in experiments to identify protein partners of KDM4A that involved in site-specific copy number gain in tumors, specifically at the 1q12h region. Expression constructs were transfected into HEK293T cells using the FuGENE^® HD Transfection Reagent. The HaloTag-KDM4A (Cat.# FHC00602) and HaloTag-Suv39h1 (Cat.# FHC09879) expression constructs were obtained from the Kazusa DNA Research Institute (Kisarazu, Japan). Interacting proteins identified included DNA polymerase subunits and members of the minichromosome maintenance (MCM) complex. (4408)

Notes: FuGENE^® HD Transfection Reagent was used to transiently transfect HeLa cells seeded on 12mm coverslips in 24-well plates. Cells were seeded at a concentration of 5 × 10⁴ cells/well and transfected after 24 hours using a 2:1 ratio of FuGENE^® HD reagent to DNA (4µl of reagent, 2µg of DNA). Transiently transfected cells were used for confocal microscopy. For luciferase assays, HEK293 cells were seeded into 48-well plates at 40% confluency, and 24 hours later, FuGENE^® HD reagent was used to transfect the cells with one of a variety of constructs. (4411)

Notes: The authors transiently transfected 5 × 10⁶ HEK293-AAV cells per 100cm dish using the FuGene® HD Transfection Reagent, 9µg of DNA and a reagent:DNA ratio of 3:1. (4431)

Notes: These authors identified a previously unknown interaction between the transcription factor HIF1A and the cyclin-dependent kinase CDK8 (a component of the Mediator complex) in the regulation of genes associated with cellular survival under low-oxygen conditions. As part of the study, HaloTag technology was used to identify proteins interacting with CDK8 in a colorectal cancer cell line. Specifically, cells were transfected with CDK8 and CDK19 HaloTag fusion constructs obtained from Kazusa Institute. The cell lysates were then used in pulldown assays to capture interacting proteins. The results showed that CDK8 and CDK19 are present in mutually exclusive Mediator complexes. Details of the transfection are as follows: HCT116 cells were plated in 150 mm dishes and grown to 70%–80% confluence before transfection with 30 μg of plasmid DNA using FuGENE HD Transfection Reagent. (4355)

Notes: In this paper, the FuGENE^® HD Transfection Reagent was used to transiently transfect Vero, Hep-2 and HeLa cells. Transfection conditions were as follows: Vero cells plated in 60mm dishes at 4 × 10⁵ cells per plate and transfected with 3.5µg of DNA; Vero cells plated on 24-well plates at 5 × 10⁴ cells/well and transfected with 1µg DNA; Hep-2 cells plated in 12-well plates at 5 × 10⁴ cells/well on glass cover slips and transfected with 1µg of DNA; Hep-2 cells plated in 24-well plates at 5 × 10⁴ cells/well on glass cover slips and transfected with 0.5µg and 1µg of DNA; HeLa cells plated in 24-well plates at 5 × 10⁴ cells/well and transfected with 1µg of DNA. A 3:1 ratio of reagent to DNA was used for all transfections. (4419)

Notes: The authors transiently transfected 5 × 10⁶ HEK293 cells per 150cm dish using the FuGENE® HD Transfection Reagent, 15µg of DNA and a reagent:DNA ratio of 7:2. (4432)

Notes: HepG2 cells were plated, 5 × 106 cells in 15cm plates. Transfections were performed 24 h later using Fugene HD. The authors used 15 μg of DNA per transfection and a Fugene:DNA ratio of 7:2. (4425)

Notes: These authors set out to determine how TET2 and TET3 proteins are involved in epigenetic regulation. To characterize proteins that interact with TET, the authors expressed full-length TET1, TET2 and TET3 as HaloTag® fusion proteins and performed protein pull-downs. They identified novel interactions between all three TET proteins and O-GlcNAc transferase (OGT), which catalyzes the addition of N-acetylglucosamine (GlcNAc) to numerous transcription factors, regulatory proteins and histones to activate or inhibit the target protein or recruit additional proteins. In this paper, they focused on TET2 and TET3, which showed the strongest interaction with OGT. They mapped TET2, TET3 and OGT binding throughout  the genome by expressing these proteins as HaloTag® fusion proteins in HEK293T cells, crosslinking the proteins and DNA, then capturing the fusion proteins and associated DNA fragments and performing high-throughput sequencing to show that TET2/3 and OGT colocalize at active gene promoters and were tightly clustered near transcription start sites.

For expression of HaloTag® fusion proteins and controls, HEK-293 cells were plated at 12 ×10⁶ cells in a 150mm dish and grown to 70–80% confluency before transfection with 30µg of plasmid using the FuGENE® HD Transfection Reagent.

To assess whether TET2/3-OGT activity affects the interaction of SET1/COMPASS with chromatin, the authors used bioluminescence resonance energy transfer (BRET). They created a fusion protein consisting of the H3K4 methyltransferase SETD1A and NanoLuc® luciferase as the energy donor and a fluorescently labeled histone H3.3-HaloTag® fusion protein as the energy acceptor.  These BRET experiments confirmed that TET2/3-OGT activity is necessary for SET1/COMPASS complex function and showed that TET and OGT activities promote binding of SETD1A, a component of the SET1/COMPASS complex, to chromatin. This binding increases H3K4me3 levels. Thus, the authors’ data support a TET2/3-OGT-mediated mechanism for regulating the SET1/COMPASS complex and thus H3K4me3. (4262)

Notes: FuGENE HD reagent was used for transient transfection of A549 cells and wild type mouse embryonic fibroblasts. A 3:1 ratio of reagent to DNA was used in the transfection reactions (9µl reagent, 3µg DNA). (4410)

Notes: To better understand what roles FAM123A may play in signaling, cell behavior and human disease, HT1080 sarcoma cells were plated on MatTek dishes coated with 5mg/ml fibronectin before transfection with Venus (a yellow fluorescent protein)-tagged FAM123A or Venus-WTX, another member of the FAM123 gene family, using FuGENE® HD Transfection Reagent. The cells were imaged the next day for low-resolution analysis. For a higher magnification, dynamic analysis, HT1080 cells were plated onto Delta T dishes coated with 5mg/ml fibronectin and transfected with EGFP-FAM123A and FuGENE® HD Transfection Reagent. The next day, cell images were captured every 10 seconds. Examining the effect of silencing FAM123A, a reporter assay used the pGL4.34[luc2P/SRFRE/Hygro] Vector cotransfected with a CMV-Renilla coreporter and other effector plasmids or siRNA, and luciferase levels assessed using the Dual-Glo® Luciferase Assay System. (4246)

Notes: Fugene HD was used with HeLa-TetOff cells, seeded at a density of 100,000 cells/well of a 12-well culture plate. On day 2, cells were transfected with 0.5 μg total DNA/well and FuGene HD reagent with a DNA to FuGENE HD ratio of 1:3 (w/v). (4423)