*, < 0.05; **, TAK-242 S enantiomer < 0.01; ***, < 0.001. treatment of YAP-expressing cells induced cell death due to Mcl-1 depletion. In a YAP-associated mouse model of CCA, expression of FGFR 1, 2, and 4 was also significantly increased. Accordingly, BGJ398 treatment was tumor-suppressive in this model and in a YAP-positive PDX model. These preclinical data suggest not only that the YAP and Hippo signaling pathways culminate in an Mcl-1-regulated tumor survival pathway but also that nuclear YAP expression may be a biomarker to employ in FGFR-directed therapy. gene fusions in solid organ malignancies including CCA (10C15% TAK-242 S enantiomer prevalence in CCA) (4, 14,C17). FGFR4 overexpression has also been associated with human CCA tumor progression and adverse survival (18). These observations raise the specter that deregulated FGFR expression and signaling also play a critical role in CCA biology. FGFRs are transmembrane tyrosine kinases belonging to the immunoglobulin superfamily. The receptor family comprises four closely related genes, forwardreverseforwardreverseforwardreverseforwardreverseforwardreverseforwardreverseforwardreverseforwardreverseforwardreverseforwardreverseforwardreverseforwardreverseforwardreverseforwardreverseforwardreverseforwardreverseforwardreverseforwardreverseforwardreverseand subsequently removing the supernatant and resuspending the beads with wash buffer (0.1% SDS, 1% Triton X-100, 2 mm EDTA, 150 mm NaCl, and 20 mm Tris-HCl, pH 8). DNA was eluted by adding 150 l of elution buffer (1% SDS and 100 mm NaHCO3) to the A/G beads and incubating for 15 min at 30 C with rotation. The samples were spun down, TAK-242 S enantiomer and the supernatant was placed into a fresh tube. The DNA was purified using a QIAquick PCR Purification Kit (Qiagen) and used for PCR analysis. Primers set in a section of chromosome 10 that does not have any known genes, often referred to as the gene desert, TAK-242 S enantiomer were used as a negative control. Proximity Ligation Assay Cells were cultured on glass coverslips, fixed with 4% paraformaldehyde for 20 min at 37 C, and permeabilized with 0.1% Triton X-100 for 10 min at 37 C. The cells were then processed according to the manufacturer’s protocol (Duolink fluorescence, Sigma) and analyzed using a fluorescent confocal microscope equipped with an ultraviolet laser (LSM 780, Zeiss). In SPP1 Vitro Relative Cell Number and Proliferation Studies Cell viability was assessed by the MTS assay and cell proliferation by the bromodeoxyuridine (BrdU) incorporation assay. For TAK-242 S enantiomer MTS, cell lines were seeded into 96-well plates (5000 cells/well) for 24 h. Cells were then treated with either vehicle or BGJ398 for 48 h, and samples were processed according to the manufacturer’s instructions (CellTiter 96 Aqueous One Solution, Promega). Absorbance was measured at 490 nm by a microplate reader (BioTek Synergy H1). For the BrdU incorporation assay, cells were treated as described above. Samples were processed according to the manufacturer’s instructions (BrdU cell proliferation assay kit, Millipore), and absorbance was measured at 450 nm using a microplate reader (BioTek Synergy H1). Quantification of Cell Death Cells were produced to subconfluency in 96-well plates, and their respective treatments were added subsequently. Cellular nuclear morphology was assessed by fluorescent microscopy after staining with DAPI (Sigma), and apoptosis was quantified as described previously (24). KMBC wild-type cells and cells stably transfected with Mcl-1 overexpression were produced to 15C20% confluency in 96-well plates, and the respective treatments were added with a total volume of 50 l in each well. After 48 h, Sytox Green (Life Technologies) was diluted in DMEM to a concentration of 5 m. 10 l of this dilution was added to each well. Following a 15C20-min incubation, fluorescence was measured at 488 nm excitation and 520 nm emission. 10 l of 250 m digitonin was added to each well and allowed to incubate for 30 min. Fluorescence measurements were obtained again, and cell death was calculated as a percentage of this maximum induced fluorescence. Extracellular Flux Analysis of Overall Oxygen Consumption Rates (OCR) and ATP Measurements Mitochondrial respiration was decided in KMCH and KMCH-Mcl-1 cells using an XF24 extracellular flux analyzer (Seahorse Biosciences, North Billerica, MA) as described by Wu (27). Briefly, KMCH and KMCH-Mcl-1 cells were seeded in 24-well cell culture microplates.