Protein bands were detected with SuperSignal West Pico chemiluminescence substrate Palbociclib chemical structure (Pierce) and processed with the GenTools software package. In each experiment, the same amount of protein was used, and the experiments were repeated independently at least three times. Chromatin immunoprecipitation (ChIP) assays ChIP assays were performed using a ChIP Assay Kit (Upstate Biotechnology, Lake Placid, NY, USA) on A549 cells cultured to 70-80% confluence. Chromatin was cross-linked with 1% formaldehyde at 37°C for 10 min. Cells were washed with cold PBS twice and disrupted in

SDS lysis buffer containing protein inhibitor cocktail. Chromatin was sonicated to an average length of 200 to 1000 bp as verified by agarose gel. Sonicated cell supernatants were diluted 10-fold in ChIP dilution buffer containing protein inhibitor cocktail and an aliquot was reserved for input control. Antibody against c-Myc (10 μg, Abcam, Cambridge, MA) was added and the chromatin solution was gently rotated overnight on ice. Protein A agarose slurry was added and

incubated at 4°C for 1 h with constant rotation. Agarose beads compound screening assay were collected by centrifugation and washed, and antibody-bound chromatin released from the agarose beads. DNA was purified by phenol/chloroform extraction and ethanol precipitation. Binding was detected by PCR. A 10-kb region downstream from the binding site was used as a negative control. shRNA transfection ShRNA constructs against c-Myc, eIF4E and CDK4 were from Origene Company (Rockville, MD). A549 or H23 cells were cultured until 70%-80% confluence. Cells were transfected with shRNA using transfection reagent Fugene HD (Roche) according to the

manufacturer’s instructions. The level of miR-145 expression was determined using PCR. Statistical analysis All data are presented as mean ± standard deviation (SD). Statistical significance was determined by two-tailed Student’s t -test. P -values of < 0.05 were considered statistically significant. Analyses used GraphPad Prism version 5.0 for Windows, GraphPad Software (San Diego, CA). Results Expression profile of miR-145 in non-small cell lung cancers Prompted by numerous reports of miR-145 downregulation in cancer [25–27], we sought to identify the role of miR-145 in NSCLC. We compared the expression levels of miR-145 in NSCLC compared to corresponding GPX6 normal tissues by qPCR for miR-145 in 37 matched pairs of tumor and non-tumor tissues from patients. We also measured expression in a non-tumorigenic lung cell line and two human NSCLC cell lines. As shown in Figure 1A, miR-145 expression levels were significantly decreased in tumors compared to the paired normal samples. Further, compared to the normal lung cell line Gekko Lung-1, the NSCLC cell line A549 showed about 80% significantly lower expression of miR-145, and H23 NSCLC cells showed approximately 50% lower expression (Figure 1B).