After being incubated with appropriate secondary antibodies, the antigen-antibody complexes were detected by using an ECL blotting analysis system (Amersham Pharmacia Biotech, Piscataway, NJ, USA). subsequently reduces a cancer stem-like cell population in human lung cancer cells. Our data suggest that CK2 inhibitors may be beneficial to the lung cancer patients with activated Notch1 signalling. was assessed using a CellTiter-Glo Luminescent cell viability assay (Promega Corporation, Madison, WI, USA), according to the manufacturer’s protocol 21. Tissue samples and immunohistochemistry Fresh lung cancer tissues were obtained from patients with lung cancer who were undergoing surgical resection of the primary tumour. All human tissue samples were obtained and analysed in accordance with procedures approved by the institutional review board of the University of California, San Francisco (IRB H8714-22 942-01). We obtained written informed consents from all participants lithospermic acid involved in our study. The tissue microarray sections were immunostained as previously described 21. Anti-Notch1 antibody was from Cell Signalling (Beverly, MA, USA; D1E11). The following scoring system was employed: ?, no stain; +, weak staining (30% or above stained cellularity considered as positive); ++, moderate staining (10% or above stained cellularity considered as positive); +++, strong staining (positive). All scoring systems were under low magnification (10 ). siRNA and plasmid DNA transfection CK2 siRNA (ON-TARGET SMARTpool) and control siRNA were purchased from Thermo Scientific (Waltham, MA, USA). In brief, cells were seeded in a 6-well plate as 105 cells/well lithospermic acid 1 day before transfection, with a target of 30C50% confluency at the time of transfection. Cells were transfected with 50 nmol/l of siRNA using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. lithospermic acid Adequate inhibition of the siRNA-mediated knockdown was confirmed by Western blot. The pcDNA3.1-CK2 or control pcDNA3.1-LacZ plasmid vectors were then transfected into the A549 cells (0.5 g/ml in 24-well plate) lithospermic acid using Lipofectamine 2000 transfection reagent (Invitrogen), according to the manufacturer’s protocol. Cells were harvested for RT-PCR and Western blot or used in reporter assays at 48 hrs post-transfection. RNA isolation, cDNA synthesis and semi-quantitative RT-PCR Isolation of RNA was performed using RNeasy Mini kit (Qiagen, Valencia, CA, USA). Normal human lung total RNA was IL6R purchased from Clontech Laboratories (Cat. #: 636524, Mountain View, CA, USA). The normal lung sample was pooled from three Caucasians without lung cancer (aged from 32 to 61). Five-hundred nanogram of total RNA was converted into 20 l cDNA using iScript cDNA Synthesis Kits (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s recommendations. PCR bands were visualized under UV light and photographed. Real-time-PCR A total of 2 l of the reverse transcription reaction mixture were used as template for real-time detection using TaqMan Technology on an Applied Biosystems 7000 sequence detection system (Applied Biosystems, Foster City, CA, USA). Gene expression was quantified for the tested genes and endogenous control gene b-glucuronidase (GUSB) using the primer and probe sequences commercially (Applied Biosystems). Western blot analysis Whole protein was extracted by M-PER Mammalian Protein Extraction Reagent (Thermo Scientific) from cell lines added with Phosphatase Inhibitor Cocktail Set II (Calbiochem, San Diego, CA, USA) and lithospermic acid Complete Protease Inhibitor Cocktails (Roche, Lewes, UK) according to manufactures’ protocols. The proteins were separated on 4C15% gradient SDSCpolyacrylamide gels and transferred to Immobilon-P membranes (Millipore, Bellerica, MA, USA). The following primary antibodies were used: anti-CK2 (Millipore), anti-Notch1 (Cell Signalling), anti-Hes1 (BD Biosciences, San Jose, CA, USA) and anti-GAPDH (Trevigen, Gaithersburg, MD, USA). After being incubated with appropriate secondary antibodies, the.