Figure 4.

EMX2 suppressed canonical WNT signaling in lung cancer cells. (a) Quantitative RT–PCR of EMX2 expression in cell lines stably transfected with control or EMX2 expression vector (in H1299) and with non-silencing control or EMX2-specific shRNA (in A427). H1703 served as an EMX2 expression level control. (b, c) TOP/FOP luciferase assays (performed 24 h after transfection as previously described in Clement et al., 2008) in H1299 cells stably transfected with EMX2 cDNA and in A427 cells stably transfected with EMX2 shRNA, respectively. (d) Western blotting of key canonical WNT downstream effector (cytosolic β-catenin; antibody from BD Biosciences) and target protein (Cyclin D1; antibody from Cell Signaling Technology, Danvers, MA, USA). β-Actin (antibody from Sigma) was used as protein control. Cytosolic proteins were prepared by using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce Biotechnology, Rockford, IL, USA) according to the manufacturer’s protocol. (e) The effect of DAC on key canonical WNT downstream effectors. Semiquantitative RT–PCR was used to confirm reactivation of the EMX2 expression after treatment of cells lines with 10 μM DAC. DAC treatment and semiquantitative RT–PCR were performed as described in Figure 1e. GAPDH served as control for RNA quality and loading. Western blotting of cytosolic β-catenin and Cyclin D1 was performed as described in Figure 4d. β-Actin was used as protein control. (f) Microarray profiling of lung cancer cell line H1299 stably transfected with EMX2 and empty vector control. Partial heatmap plot of hierarchical clustering including S100A4 and S100P is shown. Total RNA quality was assessed using a Pico Chip on an Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). RNA was amplified and labeled with Cy3-CTP or Cy5-CTP using the Agilent low RNA input fluorescent linear amplification kits following the manufacturer’s protocol. Labeled cRNA was assessed using the Nandrop ND-100, and equal amounts of Cy3- and Cy5-labeled target were hybridized to Agilent whole-human genome 44 K ink-jet arrays. Hybridization samples were randomized on the 3 × 44K format to correct any batch bias. Hybridizations were performed for 14 h according to the manufacturer’s protocol. Arrays were scanned using the Agilent microarray scanner and raw signal intensities were extracted with Feature Extraction v9.5 software (Agilent).