Notes: The authors investigated the role of the liver kinase B1 (LKB1)-adenosine monophosphate-activated kinase (AMPK) signaling pathway in cancer cell survival and metabolic adaptation. The CellTiter-Glo® 2.0, ROS-Glo™ and CellTiter® 96 AQueous One Solution Assays were used to monitor ATP content, reactive oxygen species and cell proliferation in LKB1 and AMPK knockdown cells. Additionally, the pGL3 Vector and Dual-Luciferase® Assay System were used to monitor matrix metalloproteinase-9 (MMP-9) promoter activity. (5162)

Notes: These authors studied the role of the antioxidant transcription factor nuclear factor E2-related factor-2 (Nrf2) in adaptation to inflammatory/environmental stress in malignant colonic epithelial cells. They used the Dual-Glo® Luciferase Assay and pGL3-Basic Vector in reporter assays, and the CellTiter® 96, Caspase-Glo® 3/7 and Glucose Uptake-Glo™ Assays to investigate Nrf2 effects on cell viability and metabolism. They found that Nrf2 has an impact on the metabolism in premalignant colonic epithelial cells exposed to inflammatory M1 macrophages, an effect accompanied by growth and survival alterations.  (5014)

Notes: Researchers evaluated the impact of inhibiting autophagy on cells in vitro using cell viability, cytotoxicity and caspase activity assays. (5033)

Notes: In this study, the viability of islet cell populations was evaluated using the CellTiter® 96 AQueous ONE Solution, and apoptosis levels assessed using the Caspase-Glo® 3/7 Assay. (5046)

Notes: These authors investigated whether localized bacterial infection could be used to help stimulate an immune response and render prostate tumors susceptible to immune checkpoint inhibitors. They showed that an E.coli strain (CP-1) isolated from a prostate infection colonized prostate tumors and enhanced immune checkpoint inhibition. The Caspase-Glo® 3/7 Assay, CellTiter® AQueous ONE Solution and the CytoTox® 96 Assay were used to assess apoptosis, cell viability and cytotoxicity, respectively.  (5047)

Notes: Researchers were interested in detecting Helicobacter pylori infecting C57BL/6 mice in vivo using a fluorescent probe specific for the 16S rRNA gene. To test if the probe, Cy3_HP_ LNA/2OMe _PS, was cytotoxic to cells, the human gastric epithelial cell line AGS was seeded in a 96-well plate at 1.6 × 10⁵ cells/well and the next day, medium changed and 0.4–2 µM of probe or vehicle only was added to the AGS cells. After 24 hours, cell viability was determined using the CellTilter 96® Aqueous One Solution Cell Proliferation Assay. The potential genotoxicity of the H. pylori-specific probe was assessed using the VITOTOX® Assay, and the luminescent signal measured every 5 minutes over 4 hours using the GloMax® Discover System. (4749)

Notes: A combination therapy of the chemotherapeutic cisplatin and an IL-1R antagonist was tested as a potential treatment in malignant mesothelioma (MM). The authors observed both caspase-1 activation and pyroptosis upon treatment. Caspase activation was measured using the Caspase-Glo® 1 assay, and pyroptosis was measured with the CellTiter® 96 Aqueous One Solution Assay. (5148)

Notes: The effect of overexpression of MAP2K1 (D67N) was assessed in the Calu-1 human cells through transfection with ViaFect™ Transfection Reagent (details not provided). The effect of 72 hour cisplatin exposure on 13 different human lung cell lines was assessed with the CellTiter 96® AQueous One Solution Assay. All cells were authenticated through STR analysis using the GenePrint® 10 System. (4682)

Notes: Total RNA was isolated from untreated HepG2 cells to clone the AS3MT transcript by RT-PCR, and for RT-qPCR analysis following AS3MT-directed siRNA treatment. The total RNA was isolated with the ReliaPrep™ RNA Cell Miniprep System. The health of the siRNA-treated cells was monitored with the CellTiter 96® AQueous One Solution Cell Proliferation Assay. (4620)

Notes: The authors used the CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) as a third assay (in addition to ³H thymidine and ³H leucine assays) to assess the response of cells to CoArgIPEG and demonstrated not only a decline in cell survival with treatment but also a decline in proliferation and protein synthesis in most cases.

The CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) was also used to evaluate the effect of recovery in cancer cells. The authors determined that prolonged treatment resulted in cytotoxicity; however, assessment at 24 hours showed that some cells survived in the short term. Because the authors knew proliferation/protein synthesis inhibition was occurring, evaluation with a cell survival assay like the CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) was required for a more complete understanding of cellular health and response. (4511)

Notes: The authors explore barley (Hordeum vulgare) as a means of expressing recombinant human Flt3 ligand, which is a growth factor involved in proliferation and differentiation of stem cells and development of various immune cells. As part of their quality control, they performed in-gel proteolytic digestion and mass spectrometry. The recombinant Flt3 ligand was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by Coomassie® blue staining, excision of the protein band, destaining, drying of the gel slice and digestion with Sequencing Grade Modified Trypsin at 30°C overnight prior to mass spectrometry. To test biological activity, the authors treated human acute myeloid leukemia cells with Flt3 expressed in barley or a commercial source of Flt3 then measured cell proliferation using the CellTiter 96® AQ_ueous One Solution Cell Proliferation Assay. (4352)

Notes: This paper examined the role of the Cdk3/c-Jun signaling in EGF-stimulated cell proliferation and cell transformation. An AP-1 luciferase reporter plasmid construct was contransfected with various expression vector combinations of Cdk3, Cdk2, c-Jun, c-Jun M63/73, Cdk3-DN and cyclin C and the phRL-SV40 Vector as a normalization control. Cells were lysed at room temperature for 30 minutes with gentle shaking and luciferase activity measured using the Dual-Luciferase^® Reporter Assay System. SaoS-2 cells transfected with a mock and Cdk3 RNAi vector were seeded at a density of 4 x 10³ in 96-well plates with 20µl of medium. After 24 hours, 20µl of CellTiter^® 96 AQ_ueous One Solution was dispensed to each well, and the plate incubated for 1 hour at 37°C. The reaction was stopped using 25µl of 10% SDS and absorbance was measured at 492 and 690nm. For the CheckMate™ Mammalian Two-Hybrid System, the plasmid constructs, pACT-c-Jun, pBIND-Cdk3 and pG5luc, were cotransfected into HEK293 cells at no more than 100ng/well in a 48-well plate. After incubation and lysis, 20µl of lysate was dispensed into each well of a 96-well luminescence plate. Firefly and Renilla luciferase activity was detected. (4028)

Notes: The authors examined the role of p53 in modulating dUTPase promoter activity. Base substitution mutations of Sp1- and E2F-binding sites in the dUTPase promoter were performed using the GeneEditor™ in vitro Site-Directed Mutagenesis System. Each mutant was confirmed by DNA sequencing. To determine growth inhibition, HCT116 human colon cancer cells were seeded in 96-well plates at 3 × 10³ cells/well and treated with 5-fluorouracil (5-FU), fluorodeoxyuridine (FUdR), oxaliplatin or in combination. After 72 hours, the CellTiter^® 96 AQ_ueous One Solution was dispensed into each well and absorbance measured. RNA was isolated from HCT116 p53+/+ and HCT116 p53-/- cells. cDNA was reverse transcribed from 200ng total RNA followed by multiplex qPCR using the Plexor™ qPCR System to amplify dUTPase, thymidylate synthase and GAPDH, a housekeeping gene. The 1.2 kb region of the dUTPase promoter upstream of the transcriptional start site was amplified by PCR and the fragment cloned into the pGL3-Basic Vector. Truncated promoters were also generated by PCR and cloned into the same vector. Drosophila SL-2 cells and HCT116 cell lines were seeded in a 24-well plate and transfected with dUTPase pGL3 promoter constructs or with pCI-Neo:p53WT, pCI-Neo:p53MUT and the empty pCI-neo Mammalian Expression Vector; all transfections included the pRL-TK Vector at a ratio of 1:10. After six hours, the cells were incubated in either fresh medium or medium containing a cytotoxic agent at the appropriate concentration. Thirty hours later, the cells were lysed, quantitated by Western blotting and 20µl of lysate analyzed with the Dual-Luciferase^® Reporter Assay System. Electrophoretic mobility shift analyses (EMSA) were performed using –64 to –91 of the dUTPase-nuclear isoform transcriptional start site in the Gel Shift Assay System. (4031)

Notes: To examine the role of cyclin-dependent kinase (cdk)-3 expression in cancer cell lines, potential targets of cdk3 phosphorylation were examined. The CheckMate™ Mammalian Two-Hybrid System was used to test for transcription factor binding partners of cdk in HEK293 cells. Cell proliferation of T96G cells stably transfected with either cdk3 or vector was assessed using the CellTiter 96® AQueous One Solution Cell Proliferation Assay. (3983)

Notes: The authors of this study investigated the relationship of somatic mutations that deregulate the RAS-RAF-MEK-ERK pathway and Acute Lymphoblastic Leukemia (ALL) and its progression to relapse in some patients. They show that such mutations are common in ALL and its recurrence. Furthermore, lymphoblasts from patients with mutations that were associated with upregulation of ERK showed increased cytotoxicity over wild-type controls when treated with U0126, suggesting that specific ERK inhibitors may eventually yield useful therapeutics. Following drug exposure, cytotoxic effects were assessed using the CellTiter 96® AQ_ueous One Solution Cell Proliferation Assay. (3905)

Notes: The authors examined the role of ceramide and NF-κB in age-related adipose tissue inflammation in mice. Levels of inflammation-associated molecules, IL-1β, IL-6, TNF-α, COX-2 and peroxisome proliferator-activated receptor (PPAR)-γ mRNA, were quantified by quantitive PCR (qPCR). RNA was isolated from adipose tissues, and first-strand cDNA was synthesized using the Reverse Transcription System prior to qPCR. mRNA levels were also quantified in peritoneal macrophages grown in the presence of young adipocyte-conditioned medium and old adipocyte-conditioned medium. Viability of the primary adipocytes used in these experiments was confirmed using the CellTiter 96® AQueous One Solution Cell Proliferation Assay and CytoTox 96® Non-Radioactive Cytotoxicity Assay. (3777)

Notes: Heregulin is a growth factor that binds to the HER/ErbB family of receptors, which includes four HER members. HER1 and HER2 overexpression in breast cancer cells is associated with higher malignancy and poorer prognosis. However, HER4 expression is associated with longer survival and more positive prognosis. The authors of this study investigated HER4-dependent growth inhibition that is mediated by heregulin. The authors determined the absolute levels of HER4 mRNA in several human breast cancer and mouse cancer cell lines. For this determination, relative cell numbers were determined using a CellTiter® AQ_ueous MTS assay. Apoptosis in heregulin-treated or untreated cells was also determined using the DeadEnd™ Colorimetric TUNEL System. The authors concluded that heregulin decreases growth of HER4-positive breast cancer cells, and that this growth inhibition is dependent on BRCA1. (3596)

Notes: To examine the role that BAF57, a transcriptional modulator of the estrogen receptor (ER), may have in breast cancer, BT549 cells were transfected with a reporter vector (pGL3-Basic with two estrogen response elements and the human pS2 promoter), the control pRL-CMV Vector and combinations of the following expression vectors: mERα or hERβ, BAF57, SRC1e, SRC1a and RAC3. After 16 hours, the cells were treated with 17β-estradiol. Twenty-four hours later, the cells were harvested and the luciferase activities assayed using the Dual-Luciferase® Reporter Assay System. GST-BAF57 (full-length, N- or C-domain) fusion protein was bound to Sepharose beads and incubated with 17β-estradiol or vehicle and wildtype or one of various mERα interaction domain mutants, which have been expressed and labeled with ³⁵S methionine using a TNT® rabbit reticulocyte lysate system. The beads were washed and analyzed for bound protein. ZR-75-1 cells were transfected with BAF57 siRNA then treated with 17β-estradiol 24 hours later. Cell proliferation was measured using the CellTiter® 96 AQ_ueous One Solution Cell Proliferation Assay. (3599)

Notes: The authors sought to determine the mechanism by which first-trimester trophoblasts resist FAS ligand-induced apoptosis but remain sensitive to TNFα-mediated apoptosis. First trimester trophoblasts express XAF1 [X-linked inhibitor of apoptosis (XIAP)-associated factor 1], which may be involved in regulating their response to proapoptotic signals. The authors created HaloTag™-XAF1 fusion constructs and transiently transfected the first trimester trophoblast cell line (3A). Cells were labeled with the HaloTag™ TMR ligand, and XAF1 was shown to localize to the cytoplasm. 3A cells transiently transfected with the fusion construct were also separated into cytoplasmic and mitochondrial fractions. The fractions were labeled with HaloTag™ TMR ligand. Expression of the fusion peaked at 48 hours after transfection in both mitochondrial and cytoplasmic fractions. TNFα-treatment of 3A cells induced translocation of endogenous XAF1 to the mitochondria. The authors used the Caspase-Glo® Assays to demonstrate activation of caspase-3 and caspase-9 in response to expression of XAF-1. They also show that caspase-3 activation and XIAP cleavage correlate with translocation of endogenous XAF1 to mitochondria. Viability of 3A and primary trophoblasts over expressing XAF1 was evaluated using the CellTiter® 96 AQueous One-Solution Assay. (3760)

Notes: The researchers sought to determine whether PPARγ expression might direct the effects of gastrin in proliferation of colorectal cancer cells (CRC). They determined that cell line DLD-1 cells had both PPARγ and gastrin receptors. They demonstrated that gastrin stimulated CRC cell proliferation with a coincident decrease in PPARγ levels. These studies show that gastrins trophic properties could be due in part to transactivation of EGFR and phosphorylation of ERK1/2, leading to a decrease in PPARγ activation.

The authors used CellTiter 96® Aqueous One Solution Cell Proliferation Assay to measure cell growth of the CRC cell line DLD-1.

The DLD-1 cells were transiently transfected with a luciferase vector, and FuGENE® 6 Transfection Reagent was used at a DNA ratio of 3:1 in 24-well plates. To normalize for transfection efficiency, the cells were co-transfected with a β-Gal reporter construct. The Dual-Luciferase® Assay System was used to measure PPARgamma activity with values then normalized to Beta-Gal, measured with the β-Galactosidase Enzyme Assay System.  (4280)

Notes: Uses CellTiter 96 Aqueous One Solution for cell viability determination (3313)

Notes: To examine the influence of cyclin E overexpression on gene expression in breast cancer, the breast cancer cell line MDA-MB-468 was stably transfected with a cyclin E-GFP fusion construct or GFP alone. RNA from two of these clones was isolated and pooled prior to reverse transcription and labeling. The microarrays were filtered and used in GoMiner analysis. Attachment assays were also performed with the stable cell lines. In these assays, the cells were allowed to adhere to 96-well plates for 1 hour, washed with PBS and then incubated with the CellTiter 96® AQ_ueous One Solution Cell Proliferation Assay for 1.5 hours. Absorbance was used to measure the number of attached cells. (3388)

Notes: Ba/F3 cells were transfected with constructs encoding either wildtype or mutant (Imatinib-resistant) Bcr-Abl kinase. Src-family kinase activity was assayed using the SignaTECT® Protein Tyrosine Kinase Peptide 2 and SAM® Biotin Capture Membranes. Cell viability was measured using the CellTiter 96® AQ_ueous One Solution Cell Proliferation Assay. (3405)

Notes: HUVECs were infected with adenoviruses encoding either GFP, forkhead transcription factor (FKHR), or FKHR-TM for 24, 32, or 45 hours. At each time point, the relative number of living cells was determined by the CellTiter 96® AQueous One Solution Cell Proliferation Assay. (3155)

Notes: Aliquots of 1.8 x 10³ cells were placed in 96-well microtiter plates and the cells were allowed to adhere for 6 hours before medium containing two-fold serial dilutions of Aspergillus niger-derived antibody from 20 to 0.078 g/ml (final concentrations of 10 to 0.039 g/ml) or medium alone then were added to the wells. After 72 hours incubation, relative proliferation was measured by using the CellTiter 96® AQueous One Solution Cell Proliferation Assay (3153)