Fifty micrograms of total protein was separated on sodium dodecyl sulfate–polyacrylamide gels and transferred onto nitrocellulose membranes click here (Merck Millipore, Billerica, MA). The blots were probed with antibodies against

YAP, phospho-YAP (pYAP), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Cell Signaling Technology) as well as TEAD1 (Santa Cruz Biotechnology, Dallas, TX). Secondary, HRP-coupled antibodies directed against rabbit or mouse IgG, respectively, were purchased from Cell Signaling Technology. Detection was performed as previously described [13]. A total of 100 ng of total RNA, isolated using the RNeasy Kit (Qiagen, Hilden, Germany) following the standard procedure as recommended by the manufacturer, was subjected to a single round of in vitro transcription and biotin labeling (Illumina TotalPrep RNA Amplification Kit; Ambion, Austin, TX). The resulting complementary RNA was hybridized on HumanHT-12 v4.0 Expression BeadArrays (Illumina, San Diego, CA) according to the manufacturer’s protocols using an automated liquid handling pipeline and scanned on an iScan System. Expression data were exported as unnormalized sample and control probe profiles from the Illumina GenomeStudio software and analyzed using R/Bioconductor and limma. Data were quality weighed, background-corrected,

quantile-normalized, log-transformed, and explored for differentially expressed genes with a false discovery rate (FDR) of 0.05 using Bayesian statistics. Sorafenib molecular weight Differential regulation of signaling pathways was performed using the signaling pathway impact analysis algorithm as previously described elsewhere [14]. For real-time reverse transcription–quantitative PCR (RT-qPCR) analysis, cells were lysed and total RNA was extracted using RNeasy Mini Kit

including an on-column Amylase DNase digestion step (both Qiagen). RNA was reverse transcribed using random primers and the High Capacity cDNA Reverse Transcription Kit (Life Technologies) according to the manufacturer’s specifications. Real-time qPCR for human YAP, endothelin-1 (EDN1), EDN2, V-myc myelocytomatosis viral oncogene homolog (avian) (MYC), cadherin-6 (CDH6), cysteine-rich, angiogenic inducer, 61 (CYR61), thrombospondin-1 (THBS1), and growth arrest and DNA damage-inducible, beta was performed using the SYBR Premix Ex Taq (Tli RNase H Plus) Kit (Takara Bio Europe, Saint-Germain-en-Laye, France) on a Reaplex2 Mastercycler Real-Time PCR System (Eppendorf, Hamburg, Germany). Relative fold expression levels were determined using the 2(− ΔΔCt) method [15], with GAPDH used as a housekeeping control. TEAD1-binding sites found in 5 kb upstream of the transcription start site of the indicated genes were considered and primer pairs for qPCR measurement of immunoprecipitated promoter fragments flanking the putative binding regions were designed.