Journal: PLoS ONE

Article Title: Nicotine Activates YAP1 through nAChRs Mediated Signaling in Esophageal Squamous Cell Cancer (ESCC)

doi: 10.1371/journal.pone.0090836

Figure Lengend Snippet: A. Cell growth curve measured with MTT assay indicated siRNA mediated knockdown of CHRNA3 increased the growth rate of KYSE510 cells. B. Transwell assay indicated that knockdown of CHRNA3 increased the invasion and migration of KYSE30 cells. C. Western blot analysis indicated 3 Stealth siRNA constructs effectively knocked down CHRNA3. Depletion of CHRNA3 led to a decrease of phosphorylated YAP1 and an increased of dephosphorylated YAP1, particularly a decreased S127 phosphorylation of YAP1. D. Observation of YAP1 subcellular localization with confocal fluorescence microscopy following depletion of CHRNA3. Translocation of YAP1 (green) from the cytoplasm to the nucleus was observed after siRNA mediated knockdown of CHRNA3 in KYSE510 cells for 48 h. E. Real-time PCR test indicated YAP1 targeted genes were induced by CHRNA3 knockdown in KYSE510 cell.

Article Snippet: Rabbit YAP1 antibody, CHRNA3 antibody were purchased from Proteintech Gourp (Chicago, IL 60612, USA), mouse YAP (63.7), GAPDH antibody were purchased from Santa Cruz biotechnology (Dallas, Texas 75220U.S.A.), Phospho-YAP (Ser127) antibody and β-catenin antibody, β-actin antibody were purchased from Cell Signaling Technology (Danvers, MA 01923), CHRNB4 antibody, CHRNA5 antibody and 14-3-3 antibody were purchased from Abgent. tGFP antibody, P63 antibody, tGFP labeled CHRNA3 Tureclonevector were purchased from OriGene (Beijing, CHINA 101111), α-catenin antibody was purchased from Lifetechnologies (Carlsbad, CA 92008).

Techniques: MTT Assay, Knockdown, Transwell Assay, Migration, Western Blot, Construct, Phospho-proteomics, Fluorescence, Microscopy, Translocation Assay, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Nicotine Activates YAP1 through nAChRs Mediated Signaling in Esophageal Squamous Cell Cancer (ESCC)

doi: 10.1371/journal.pone.0090836

Figure Lengend Snippet: A. Endogenous IP test showed positive protein interactions between YAP1 and CHRNA3/CHRNB4/CHRNA5 in KYSE510 cells. B. Positive interaction between tGFP-CHRNA3 and YAP1 was detected in cells transfected with tGFP-CHRNA3. C. GST Pull assay indicated positive interaction of exogenously expressed GST-YAP1 with CHRNA3 in KYSE510 cell. D. Confocal immunofluorescence microscopy observed colocalization of YAP1 and CHRNA3 in KYSE510 cell. The KYSE510 cells were transiently expressed tGFP labeled CHRNA3 of TureClone vector (OriGene). YAP1 was labeled with red-FITC conjugated goat anti rabbit IgG. Cell nuclei were visualized with DAPI. E. endogenous IP detected positive interactions between 14-3-3 and CHRNA3/CHRNB4/CHRNA5.

Article Snippet: Rabbit YAP1 antibody, CHRNA3 antibody were purchased from Proteintech Gourp (Chicago, IL 60612, USA), mouse YAP (63.7), GAPDH antibody were purchased from Santa Cruz biotechnology (Dallas, Texas 75220U.S.A.), Phospho-YAP (Ser127) antibody and β-catenin antibody, β-actin antibody were purchased from Cell Signaling Technology (Danvers, MA 01923), CHRNB4 antibody, CHRNA5 antibody and 14-3-3 antibody were purchased from Abgent. tGFP antibody, P63 antibody, tGFP labeled CHRNA3 Tureclonevector were purchased from OriGene (Beijing, CHINA 101111), α-catenin antibody was purchased from Lifetechnologies (Carlsbad, CA 92008).

Techniques: Transfection, Immunofluorescence, Microscopy, Labeling, Plasmid Preparation

Journal: PLoS ONE

Article Title: Nicotine Activates YAP1 through nAChRs Mediated Signaling in Esophageal Squamous Cell Cancer (ESCC)

doi: 10.1371/journal.pone.0090836

Figure Lengend Snippet: A. Nicotine administration stimulates the growth of esophageal cancer KYSE510 cell measured by E-Plateofx CELLigence RTCA MP system. B. Nicotine administration increased the invasion and migration of esophageal cancer KYSE30 cells in transwell assays. C. Subcellular localization of YAP1 examined with confocal fluorescence microscope. Translocation of YAP1 (green) from the cytoplasm to the nucleus was observed after nicotine administration in KYSE510 cells for 48 h. D. KYSE510 cells were treated with nicotine for 48 hs, decreased phosphorylation of YAP1 and increased dephosphorylated YAP1 was observed by Western blot analysis. E. Real-time PCR verification of induction of mRNAs of genes transcriptionally activated by YAP1 upon nicotine administration. F. PKC inhibitor Enzastaurin blocked nicotine induced upregualtion of YAP1 protein level, and resulted in reduction of YAP protein level, particularly the dephosphorylated form of YAP1 by Western blot.

Article Snippet: Rabbit YAP1 antibody, CHRNA3 antibody were purchased from Proteintech Gourp (Chicago, IL 60612, USA), mouse YAP (63.7), GAPDH antibody were purchased from Santa Cruz biotechnology (Dallas, Texas 75220U.S.A.), Phospho-YAP (Ser127) antibody and β-catenin antibody, β-actin antibody were purchased from Cell Signaling Technology (Danvers, MA 01923), CHRNB4 antibody, CHRNA5 antibody and 14-3-3 antibody were purchased from Abgent. tGFP antibody, P63 antibody, tGFP labeled CHRNA3 Tureclonevector were purchased from OriGene (Beijing, CHINA 101111), α-catenin antibody was purchased from Lifetechnologies (Carlsbad, CA 92008).

Techniques: Migration, Fluorescence, Microscopy, Translocation Assay, Phospho-proteomics, Western Blot, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Nicotine Activates YAP1 through nAChRs Mediated Signaling in Esophageal Squamous Cell Cancer (ESCC)

doi: 10.1371/journal.pone.0090836

Figure Lengend Snippet: A. Cell growth curve measured with MTT assay indicated siRNA mediated knockdown of CHRNA3 increased the growth rate of KYSE510 cells. B. Transwell assay indicated that knockdown of CHRNA3 increased the invasion and migration of KYSE30 cells. C. Western blot analysis indicated 3 Stealth siRNA constructs effectively knocked down CHRNA3. Depletion of CHRNA3 led to a decrease of phosphorylated YAP1 and an increased of dephosphorylated YAP1, particularly a decreased S127 phosphorylation of YAP1. D. Observation of YAP1 subcellular localization with confocal fluorescence microscopy following depletion of CHRNA3. Translocation of YAP1 (green) from the cytoplasm to the nucleus was observed after siRNA mediated knockdown of CHRNA3 in KYSE510 cells for 48 h. E. Real-time PCR test indicated YAP1 targeted genes were induced by CHRNA3 knockdown in KYSE510 cell.

Article Snippet: Rabbit YAP1 antibody, CHRNA3 antibody were purchased from Proteintech Gourp (Chicago, IL 60612, USA), mouse YAP (63.7), GAPDH antibody were purchased from Santa Cruz biotechnology (Dallas, Texas 75220U.S.A.), Phospho-YAP (Ser127) antibody and β-catenin antibody, β-actin antibody were purchased from Cell Signaling Technology (Danvers, MA 01923), CHRNB4 antibody, CHRNA5 antibody and 14-3-3 antibody were purchased from Abgent. tGFP antibody, P63 antibody, tGFP labeled CHRNA3 Tureclonevector were purchased from OriGene (Beijing, CHINA 101111), α-catenin antibody was purchased from Lifetechnologies (Carlsbad, CA 92008).

Techniques: MTT Assay, Knockdown, Transwell Assay, Migration, Western Blot, Construct, Phospho-proteomics, Fluorescence, Microscopy, Translocation Assay, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Nicotine Activates YAP1 through nAChRs Mediated Signaling in Esophageal Squamous Cell Cancer (ESCC)

doi: 10.1371/journal.pone.0090836

Figure Lengend Snippet: A. Endogenous IP test showed positive protein interactions between YAP1 and CHRNA3/CHRNB4/CHRNA5 in KYSE510 cells. B. Positive interaction between tGFP-CHRNA3 and YAP1 was detected in cells transfected with tGFP-CHRNA3. C. GST Pull assay indicated positive interaction of exogenously expressed GST-YAP1 with CHRNA3 in KYSE510 cell. D. Confocal immunofluorescence microscopy observed colocalization of YAP1 and CHRNA3 in KYSE510 cell. The KYSE510 cells were transiently expressed tGFP labeled CHRNA3 of TureClone vector (OriGene). YAP1 was labeled with red-FITC conjugated goat anti rabbit IgG. Cell nuclei were visualized with DAPI. E. endogenous IP detected positive interactions between 14-3-3 and CHRNA3/CHRNB4/CHRNA5.

Article Snippet: Rabbit YAP1 antibody, CHRNA3 antibody were purchased from Proteintech Gourp (Chicago, IL 60612, USA), mouse YAP (63.7), GAPDH antibody were purchased from Santa Cruz biotechnology (Dallas, Texas 75220U.S.A.), Phospho-YAP (Ser127) antibody and β-catenin antibody, β-actin antibody were purchased from Cell Signaling Technology (Danvers, MA 01923), CHRNB4 antibody, CHRNA5 antibody and 14-3-3 antibody were purchased from Abgent. tGFP antibody, P63 antibody, tGFP labeled CHRNA3 Tureclonevector were purchased from OriGene (Beijing, CHINA 101111), α-catenin antibody was purchased from Lifetechnologies (Carlsbad, CA 92008).

Techniques: Transfection, Immunofluorescence, Microscopy, Labeling, Plasmid Preparation

Journal: PLoS ONE

Article Title: Nicotine Activates YAP1 through nAChRs Mediated Signaling in Esophageal Squamous Cell Cancer (ESCC)

doi: 10.1371/journal.pone.0090836

Figure Lengend Snippet: B. KYSE510 cells exposed to nicotine for more than 4 days led to upregulation of total protein level of YAP1, including both the phosphorylated YAP1 and dephosphorylated YAP1 indicated by western blot analysis.

Article Snippet: Rabbit YAP1 antibody, CHRNA3 antibody were purchased from Proteintech Gourp (Chicago, IL 60612, USA), mouse YAP (63.7), GAPDH antibody were purchased from Santa Cruz biotechnology (Dallas, Texas 75220U.S.A.), Phospho-YAP (Ser127) antibody and β-catenin antibody, β-actin antibody were purchased from Cell Signaling Technology (Danvers, MA 01923), CHRNB4 antibody, CHRNA5 antibody and 14-3-3 antibody were purchased from Abgent. tGFP antibody, P63 antibody, tGFP labeled CHRNA3 Tureclonevector were purchased from OriGene (Beijing, CHINA 101111), α-catenin antibody was purchased from Lifetechnologies (Carlsbad, CA 92008).

Techniques: Western Blot

Journal: PLoS ONE

Article Title: Nicotine Activates YAP1 through nAChRs Mediated Signaling in Esophageal Squamous Cell Cancer (ESCC)

doi: 10.1371/journal.pone.0090836

Figure Lengend Snippet: The slides were scanned by the AperioScanScope CS. Tissue samples were divided into 4 categories based on the intensity of YAP1 staining.

Article Snippet: Rabbit YAP1 antibody, CHRNA3 antibody were purchased from Proteintech Gourp (Chicago, IL 60612, USA), mouse YAP (63.7), GAPDH antibody were purchased from Santa Cruz biotechnology (Dallas, Texas 75220U.S.A.), Phospho-YAP (Ser127) antibody and β-catenin antibody, β-actin antibody were purchased from Cell Signaling Technology (Danvers, MA 01923), CHRNB4 antibody, CHRNA5 antibody and 14-3-3 antibody were purchased from Abgent. tGFP antibody, P63 antibody, tGFP labeled CHRNA3 Tureclonevector were purchased from OriGene (Beijing, CHINA 101111), α-catenin antibody was purchased from Lifetechnologies (Carlsbad, CA 92008).

Techniques: Staining

Journal: PLoS ONE

Article Title: Nicotine Activates YAP1 through nAChRs Mediated Signaling in Esophageal Squamous Cell Cancer (ESCC)

doi: 10.1371/journal.pone.0090836

Figure Lengend Snippet: YAP1 expression is positively correlated with smoking status of ESCC patients 1 .

Article Snippet: Rabbit YAP1 antibody, CHRNA3 antibody were purchased from Proteintech Gourp (Chicago, IL 60612, USA), mouse YAP (63.7), GAPDH antibody were purchased from Santa Cruz biotechnology (Dallas, Texas 75220U.S.A.), Phospho-YAP (Ser127) antibody and β-catenin antibody, β-actin antibody were purchased from Cell Signaling Technology (Danvers, MA 01923), CHRNB4 antibody, CHRNA5 antibody and 14-3-3 antibody were purchased from Abgent. tGFP antibody, P63 antibody, tGFP labeled CHRNA3 Tureclonevector were purchased from OriGene (Beijing, CHINA 101111), α-catenin antibody was purchased from Lifetechnologies (Carlsbad, CA 92008).

Techniques: Expressing