Notes: In this study, researchers used the Autophagy LC3 HiBiT Reporter Assay to understand the impact of urolithin A on the autophagy pathway, using HEK293, BV2 microglia and differentiated ReNcell VM cells.  (5187)

Notes: The significant loss of estrogen in women after menopause has been linked to increased incidence of Alzheimer’s disease. Here, the protective effect of β-ecdysterone (β-Ecd) on SH-SY5Y cell apoptosis in relation to Alzheimer’s disease is investigated. NF-κB and estrogen receptor activation was monitored in the presence of β-Ecd in Aβ-stress conditions using the Dual-Glo® Luciferase Assay System. Caspase activation was measured as a proxy for cell stress and apoptosis. Together, SH-SY5Y cell treatment with β-Ecd showed a protective effect, making β-Ecd a promising therapeutic candidate. (5169)

Notes: Mucosal microbiota has been shown to influence HIV-1 infection. Here, P. gingivalis outer membrane vesicles (OMVs) show stimulation of receptor-independent HIV-1 entry into epithelial cells. A secretory NanoLuc luciferase is fused to the nef gene of the HIV genome, cell supernatant is collected, and luciferase activity is measured as a proxy for viral replication. The presence of P. gingivalis vesicles increased viral infectivity approximately 10-fold. (5107)

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: The presence of methyl-cytosine binding domain (MBD) containing proteins and ability to remodel methylated chromatin was investigate in sponges. Specifically, the sponge MBD2 and GATAD2A protein interaction was monitored in cells using the NanoBRET™ Protein-Protein Interaction System. Multiple coiled-coil fusion constructs were tested to determine optimal signal intensity. Interestingly, while DNA methylation was observed in sponges, this interaction was much lower affinity than in vertebrate organisms. (5057)

Notes: PRDM14 is dysregulated in a variety of cancers, including breast and pancreatic cancer, and overexpression leads to stem-cell-like phenotypes associated with aggressive tumors. Here, PRDM14 interacting proteins are identified using the HaloTag^® Mammalian Pull-down System. The interactions of PRDM14 and glucoseregulated protein 78 (GRP78) and heat shock protein 90-a (HSP90a) were validated in vivo with the NanoBRET™ assay. (5053)

Notes: G protein coupled receptor (GPCR) oligomerization and protein interaction has been commonly investigated using bioluminescence resonance energy transfer (BRET). The need for exogenous expression of fusion proteins has been a short coming of the BRET assay. Here CRISPR/Cas9 mediated homology directed repair was used to generate protein-NanoLuc^® fusions under endogenous expression. The GPCR- β-arrestin2 interaction serves as a model of this system, where interaction and internalization are monitored under conditions where the donor luciferase endogenously expressed. (5059)

Notes: The effect of miR-124 on the Nur77-3’ UTR was analyzed with a dual-reporter plasmid and luciferase activities were measured with the Dual-Glo® Luciferase Assay System. Daoy cells used in the study were transfected with the reporter plasmid and miRNA expression vectors using the FuGENE® 6 Transfection Reagent. FuGENE® 6 was also employed to transfect 293T cells for recombinant lentivirus construction. RNA isolation of mRNA expression level determination by probe-based RT-qPCR was accomplished with a Maxwell® 16 Research Instrument using the Maxwell® 16 LEV simplyRNA Tissue Kit. Viability assays of 2D cultures of Daoy cells were determined with the CellTiter-Glo® Cell Viability Assay. Viability assays of 3D microspheroid cultures of Daoy cells (presumably ULA plate-derived) were measured with the CellTiter-Glo® 3D Cell Viability Assay. (4816)

Notes: These authors provide an overview of the advantages and disadvantages of techniques for transfection of primary neurons, and provide an optimized protocol for FuGENE-6 transfection of primary neuronal cultures. (4720)

Notes: In this study, FuGENE® 6 was used to perform transient transfection of COS-7 cells. Plasmids containing mutated or non-mutated copies of the estrogen receptor gene ESR1 were transfected together with luciferase reporter constructs containing estrogen response elements upstream of the luciferase gene. Transactivation of the estrogen response element was reduced in the mutated estrogen receptor compared with the nonmutated  receptor. (4407)

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: In this study, MCF7 human breast adenocarcinoma cells were transfected with plasmid constructs using FuGENE^® 6 transfection reagent. Cells were grown to 50% confluence and transfected with 2µg of DNA at a 3:2 ratio of FuGENE^® 6 reagent:DNA. (4358)

Notes: RAW 264.7 mouse macrophage cells were plated in 12-well plates at 1.5 × 10⁵ cells/well for transient transfection with 0.5μg of DNA and 1.5μl of FuGENE® 6 Transfection Reagent in 50µl of Opti-MEM I medium. After 24 hours, the cells were treated with IgG IC for 5 hours and luciferase activity analyzed using the Dual-Luciferase® Reporter Assay System. (4402)

Notes: In this paper, FuGENE® 6 reagent was used to transfect PC3 human prostate cancer cells at a 4:1 FuGENE®:DNA ratio. (4370)

Notes: In this study, HEK293 cells were grown to 50-60% confluence in 100mm dishes and then transfected with plasmid constructs using FuGENE® 6 transfection reagent at a 3:1 FuGENE:DNA ratio. (4363)

Notes: In this study, FuGENE® 6 Reagent was used to transiently transfect the S2R+ drosophila Schneider cell line with 0.6µg DNA at a 8.3:1 FuGENE:DNA ratio. 0.6 x 10e6 cells were plated 1 day prior to transfection in 12-well plates. (4369)

Notes: HEK293T adenovirus-transformed human kidney cells were plated in 10cm plates with 5 × 10⁶ cells or in 12-well plates with 3 × 10⁵ cells/well before transfection using with 5µg or 0.2µg of viral reporter plasmids, respectively. To transfect the cells, FuGENE® 6 Transfection Reagent was used at a ratio of 2.5µl:1µg of reagent:DNA. (4401)

Notes: 293T cells were seeded in 12-well plates and transfected using 0.4µg of an expression plasmid, 0.6µg of a luciferase reporter plasmid and 60ng of the pRL-SV40 or pRL-TK control vector using FuGENE® 6 transfection reagent. At 24 hours posttransfection, cells were infected with Sendai virus or mouse hepatitis virus, and 8 hours postinfection, luciferase activity was measured using a dual luciferase reporter assay (Promega). (4277)

Notes: In this paper, FuGENE® 6 or HD reagent was used to transiently transfectCHO-K1 cells. Transfection conditions were as follows: Cells were plated in 60mm dishes and transfected with FuGENE® 6 using 1.03µg of DNA. Cells were plated in 100mm dishes and transfected with FuGENE® 6 or FuGENE® HD using 6µg of DNA. 

Notes: Mutations in the extracellular matrix glycoprotein cartilage oligomeric matrix protein (COMP) cause skeletal dysplasia. This study evaluated the efficacy of a ribozyme (a short catalytic RNA oligonucleotide) to knock down COMP expression. As part of the study, COS7 cells were transfected with plasmids that expressed mutant or wild-type COMP, as well as with a ribozyme targeting COMP. Transfection conditions were as follows: Cells were grown to 80%–90% confluency in 6-well plates, then transfected with plasmid DNA using a 3:2 ratio of FuGENE 6 reagent:DNA. Ribozyme treatment reduced expression of COMP mRNA in the transfected cells. (4357)

Notes: In this study, FuGENE® 6 reagent was used to transiently transfect Jurkat T lymphocyte cells and HEK 293 adenovirus-transformed  HEK 293 cells with 0.25µg DNA at a 6:1 ratio of FuGENE:DNA. The Jurkat cells were plated at 1 × 10e6 cells/plate and the HEK cells were plated at 5 × 10e5 cells/plate. (4375)

Notes: The authors examined how cytotoxicity differed between cancer cell lines treated with drugs that induced either apoptosis or paraptosis, non-apoptotic cell death. HT1080 fibrosarcoma epithelial cells were seeded in 10cm dishes and grown until 40% confluent before transfection with 15µl of FuGENE® 6 Transfection reagent and 4.5µg of wild type or mutant vector + 0.5µg of GFP vector for a 3:1 reagent-to-DNA ratio. After an eight-hour incubation, the cells were trypsinized, seeded into two 24-well plates at 100,000 cells per well), and treated with drugs 24 hours later. Sixteen hours after drug treatment, one set of plates were assessed for viability, the other, cell death. (4381)

Notes: HEK-293 were transiently transfected with adenovirus 5 DNA using FuGENE® 6 Reagent. Cells were seeded at 3 × 10⁶ cells/75 cm² and grown to 60% cell confluency prior to transfection. Cell transfections were 60–70% efficient. (4266)

Notes: HepG2 cells in a T-25 culture flask (3 million cells at 70ndash;80% confluency) were transfected with CYP3A4 reporter plasmid (a pGL3 reporter construct) and a CMV-Renilla control plasmid (a total of 2.5µg of plasmid mix was used) using FuGENE® 6 Reagent for transient transfection assays. Reporter activities were measured using the Dual-Glo® Luciferase Assay System. (4268)

Notes: Regulation of genes targeted by the ligand-activated aryl hydrocarbon receptor (AhR) has been shown to be controlled by calcium (Ca(2+)) changes induced by AhR agonists such as the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). This study investigated this link. As part of the study, MCF-7 cells were transfected with various pGL3 firefly luciferase reporter constructs and the control pRL-TK Vector expressing Renilla luciferase. Transfection conditions were as follows: MCF-7 cells were cultured in 24-well plates and transfection was performed using FuGENE® 6 transfection reagentwith a FuGENE:DNA ration of 3:1. Firefly and Renilla luciferase activities were measured using the Dual-Luciferase® Reporter Assay System. (4362)