[26] proposed that inhibition

of Gli promoted EMT in pancreatic cancers. Our study intends to extend the research to lung SCC to help us better understand the regulation of EMT by Hh signaling. We reported the activation of Hh signaling in two cohorts of patient samples, and revealed the reverse association between Gli1 expression and the expression of EMT markers. Eltanexor research buy Inhibition of the Shh/Gli pathway suppressed migration and up-regulated E-Cadherin expression in lung SCC cells. Stimulation of the pathway increased migration and down-regulated E-Cadherin expression in lung SCC cells. Materials and methods Tissue specimens Tissue specimens of the UCSF cohort were collected from 14 patients who underwent surgical resection for lung SCC at the Thoracic Oncology Program at UCSF. Tissue specimens of the Tianjin cohort were collected from 177 patients who underwent surgical resection for lung SCC at the PD0332991 in vitro Tianjin Medical University Cancer Institute and Hospital. Samples were fixed in formalin and embedded in paraffin to make tissue slides. The study with UCSF patient tissues was approved by the Committee on Human Research

(CHR approval number: H8714-11647-10) at the University of California, San Francisco (UCSF), and that with Tianjin cohort was approved by the Tianjin Medical University Cancer Institute and Hospital. Written, informed consent was obtained from each patient before specimen collection. Immunohistochemistry (IHC), immunofluorescence (IF) and Western blot Immunohistochemistry, immunofluorescence

and western blot were performed following standard procedures. Antibodies applied to detect protein expressions in IHC and IF were Gli1 (sc-20687 Santa Cruz Biotechnology, Santa Cruz, CA) at 1:100, Shh (ab 50515 Abcam, Cambridge, MA) at 1:100, Smo (ab 72130 Abcam) at 1:200, Ptch1 (Santa Oxymatrine Cruz that Biotechnology,) and E-Cadherin (EMD Millipore) Smo (Sigma, St. Louis, MO) at 1:100, E-cadherin (sc-7870, Santa Cruz Biotechnology) at 1:100, and β-catenin (BD Biosciences, San Jose, California) at 1:400. Antibodies used in Western blot were Gli (C68H3, Abcam) at 1:1000, E-Cad (HECD-1 MED Milliopore, Darmstadt, Germany) at 1:1000 and Actin (A5441, Sigma) at 1:5000. Total protein extraction was performed with M-PER Mammalian Protein Extraction Solution (Thermo Scientific, Waltham, MA), and 40ug of proteins were analyzed in Western blot. Cell culture, drug treatment and migration assay Human lung SCC cell lines H2170, H1703, H1869 and SK-MES-1 were purchased from the Cell Culture Core Facility at Harvard University (Boston, MA, USA). The cell lines were cultured in RPMI 1640 (Life Technologies, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) and antibiotics.