a-b, Immunohistochemical staining evaluation of the appearance degrees of E-cad (a) and Vim (b) in TT pancreas examples from ESs and NSs ( em n /em ?=?173). from Panc-1 cells following manufacturers guidelines, and the different truncated mutant YAP1 regulatory regions were amplified by PCR. The primers Rabbit Polyclonal to p300 used to amplify the truncated YAP1 promoter regions are shown in Supplementary Table s2. YAP1 promoter region fragments were inserted into the pGL3 vector (Promega, Madison, WI), as described previously . The YAP1 promoter reporter plasmids were co-transfected with HIF1A, or vacant vector (EV) into cells. For the RKI-1447 HIF1A transcriptional activity assay, pGL4-HREs-luciferase plasmid (4?g) and pRL-TK plasmid (4?g) were incubated with PDAC cells. These cells were also incubated with 1.0?m Nic or DMSO. After 24?h, luciferase activity was detected using the Dual-luciferase reporter assay system (Promega, Madison, WI). Chromatin immunoprecipitation (ChIP) For ChIP assay, PDAC cells were treated with 1.0?m Nic or DMSO for 12?h, as described previously . Briefly, protein-DNA complexes were produced by adding 1% formaldehyde to the cells, and the chromatin was sheared by sonication to a mean fragment size of 300C500?bp. Cells were immunoprecipitated overnight with an anti-HIF1A antibody or rabbit IgG, RKI-1447 and the associated genomic DNA was assessed by PCR and agarose gel electrophoresis. The specific primers for putative HREs in the YAP1 promoter are shown in Supplementary Table s2. Cell viability, migration and transwell assays To evaluate cell proliferation rates, MTT assay was used. Briefly, cells were seeded in 96-well plates at 2??103 cells/well and incubated with DMSO or 1.0?m Nic. Sample absorbance at 490?nm was evaluated on a microplate spectrophotometer (Thermo, Spectronic, Madison, WI, USA). For the cell scratchCwound assays, cells were cultured in six-well plates until confluent, and horizontal streaks were created in RKI-1447 the cells using the a 20-L pipette tip. Then, cells were washed and incubated with DMSO or 1.0?m Nic. An inverted microscope was used to measure the migratory distance at 0?h and 24?h, and cell migration was assessed by measuring gap sizes in multiple fields. For transwell assays, cells (1.0??10 /ml) were placed in the top side of transwell chambers (8?m pore size membranes, Millipore) with matrigel for invasion. Vehicle (DMSO) or nicotine was added into the upper well for 24?h. The invaded cells were fixed, stained and counted in five random fields. Mouse xenograft model All animal studies were performed following the Institutional Animal Care and Use Committee of Shanghai Jiaotong University (Shanghai, China). Six-week-old male BALB/c mice were obtained from Shanghai SLAC Laboratory Animal Center (Shanghai, China). All animals were maintained in a barrier facility in high-efficiency particulate airCfiltered racks. Logarithmic phase Panc-1 cells (5.0??10 /100?L) transfected with Lv-shYAP1 or control vector were inoculated subcutaneously into the dorsal flank of mice. Tumor volume was evaluated by the following formula: volume?(mm3)?=?length??width??height??0.52. When tumor volume reached 75C125?mm3, mice were randomized into three groups, and that day was defined as day 1. Nicotine or DMSO was administered intraperitoneally thrice weekly for 3?weeks. On day 22, all mice were euthanized, and the tumors were excised and weighed. Bioinformatics and statistical analysis Multiple databases, including GEPIA (http://gepia.cancer-pku.cn/) , StarBase 3.0 RKI-1447 (http://starbase.sysu.edu.cn/) , and KM plotter (http://kmplot.com/analysis), were queried for gene expression in PDAC tissues. Data are shown as mean??SD. Differences between groups were evaluated using unpaired t-test for two groups or the chi square test. Survival analyses were performed using the Kaplan-Meier method with the log-rank test and univariate and multivariate Cox regression. All statistical analyses were performed using the PASW Statistics 19.0 software program (SPSS, Chicago, IL, USA). A two tailed values of valuehazard ratio, confidence interval Tumor classification and stage were referred to the 7th edition of UICC on cancer staging system. Bold indicates significance Nicotine stimulates YAP1 nuclear translocation Nuclear localization is essential for the activity of YAP1. We decided whether nicotine treatment affects the subcellular localization of YAP1 in PDAC cells. IHC assessment indicated that YAP1 was localized in both the cytoplasm and nucleus, and mainly in the nucleus (Supplementary Fig. s5b-c). To explore the clinical significance of cytoplasmic and nuclear expression of YAP1, we evaluated associations of cytoplasmic and nuclear YAP1 expression with OS, and we found significantly shorter OS time (We also found that nicotine enhanced expression levels of YAP1 and HIF1A in a dose-dependent manner, both of which induce EMT and tumor growth in PDAC cells in vitro and in murine xenograft models. Mechanistically, nicotine induces a positive feedback loop of HIF1A and YAP1, wherein HIF1A RKI-1447 transcriptionally activates YAP1 and stimulates YAP nuclear translocation, and YAP1 increases HIF1A.