Journal: Journal of Animal Science and Biotechnology

Article Title: Kynurenine promotes porcine intestinal epithelial cell proliferation by activating the AHR-MST1-YAP1 axis

doi: 10.1186/s40104-026-01368-0

Figure Lengend Snippet: L-Trp promotes proliferation of IPEC-J2 cells. A The mRNA expression of proliferative genes PCNA , SYR and MUC13 in IPEC-J2 cells treated with different concentrations of L-Trp ( n = 6). a–c Mean values with unlike letters were significantly different ( P < 0.05). B and C EdU staining and quantification of the ratio of EdU-positive cells in IPEC-J2 cells treated with 4 mmol/L L-Trp or 5 mmol/L L-Glu. D The mRNA expression of cell proliferation associated genes Cyclin E1, Cyclin D1, TGF-β , SMAD4, MST1, YAP1 in IPEC-J2 cells after 4 mmol/L L-Trp or 5 mmol/L L-Glu treatment ( n = 6). Asterisks denote statistically significant differences ( * P < 0.05, ** P < 0.01). Error bars denote SEM

Article Snippet: For inhibiting YAP1 and AHR expression, 0.5 μmol/L Verteporfin ( HY-B0146, MedChemExpress; dissolved in 0.1% DMSO) and 0.2 μmol/L AHR inhibitor CH-223191 (HY-12684, MedChemExpress, USA; dissolved in complete medium) were co-incubated with 4 mmol/L L-kynurenine for 24 h, respectively.

Techniques: Expressing, Staining

Journal: Journal of Animal Science and Biotechnology

Article Title: Kynurenine promotes porcine intestinal epithelial cell proliferation by activating the AHR-MST1-YAP1 axis

doi: 10.1186/s40104-026-01368-0

Figure Lengend Snippet: Kyn promotes the proliferation of IPEC-J2 cells via activating the AHR-MST1-YAP1 axis. A Quantification of the mRNA expression of AHR and CYP1A1 in IPEC-J2 cells after Kyn or Kyn + Verteporfin treatment ( n = 6). B Illustration of the AHR binding sites in the promote region of the MST1 gene. C Quantification of the mRNA expression of MST1 , YAP1 , PCNA and Cyclin E1 after Kyn or Kyn + Verteporfin treatment ( n = 6). D, E EdU staining and scratch assay in IPEC-J2 cells treated with Kyn alone or Kyn + Verteporfin (Scale bar: 20 μm). F Quantification of the ratio of EdU-positive cells and the width of cell migration in IPEC-J2 cells treated with Kyn alone or Kyn + Verteporfin. G and H Quantification of the mRNA expression ( n = 6) of AHR and CYP1A1 and the protein expression ( n = 3) of PCNA in IPEC-J2 cells treated with Kyn alone or Kyn + AHR inhibitor. I Quantification of the mRNA expression of MST1 , YAP1 , PCNA and Cyclin E1 after Kyn or Kyn + AHR inhibitor treatment ( n = 6). J and K Immunostaining and scratch assay in IPEC-J2 cells treated with Kyn alone or Kyn + AHR inhibitor (Scale bar: 20 μm). The yellow arrowheads indicated the nuclear localization of AHR, while the red arrowheads indicated the cytoplasmic localization of AHR, respectively. L Quantification of the fluorescence intensity of PCNA and the width of cell migration in IPEC-J2 cells after Kyn or Kyn + AHR inhibitor treatment. Asterisks denote statistically significant differences ( * P < 0.05, ** P < 0.01). Error bars denote SEM

Article Snippet: For inhibiting YAP1 and AHR expression, 0.5 μmol/L Verteporfin ( HY-B0146, MedChemExpress; dissolved in 0.1% DMSO) and 0.2 μmol/L AHR inhibitor CH-223191 (HY-12684, MedChemExpress, USA; dissolved in complete medium) were co-incubated with 4 mmol/L L-kynurenine for 24 h, respectively.

Techniques: Expressing, Binding Assay, Staining, Wound Healing Assay, Migration, Immunostaining, Fluorescence

Journal: Journal of Animal Science and Biotechnology

Article Title: Kynurenine promotes porcine intestinal epithelial cell proliferation by activating the AHR-MST1-YAP1 axis

doi: 10.1186/s40104-026-01368-0

Figure Lengend Snippet: Kyn-induced intestinal organoids proliferation is dependent on the activation of the MST1-YAP1 pathway. A – D Representative images of porcine intestinal organoids and the quantification of budding efficiency from the control, Kyn and Kyn + Verteporfin treated groups, respectively (scale bar: 100 μm). E–H Immunostaining and the fluorescence intensity of OLFM4 in the control, Kyn and Kyn + Verteporfin treated groups, respectively (scale bar: 20 μm). I and J The protein expression of PCNA, AHR, MST1 and Cyclin D1 in intestinal organoids treated with Kyn or Kyn + Verteporfin ( n = 3). K Proposed model of Trp metabolism-derived Kyn in regulating intestinal epithelial cell proliferation via activation of the AHR-MST1-YAP axis. Trp, tryptophan; Kyn, kynurenine; AHR, aryl hydrocarbon receptor; ARNT, aryl hydrocarbon receptor nuclear translocator; ISC, intestinal stem cell; IEC, intestinal epithelial cell. Asterisks denote statistically significant differences ( * P < 0.05, ** P < 0.01). Error bars denote SEM

Article Snippet: For inhibiting YAP1 and AHR expression, 0.5 μmol/L Verteporfin ( HY-B0146, MedChemExpress; dissolved in 0.1% DMSO) and 0.2 μmol/L AHR inhibitor CH-223191 (HY-12684, MedChemExpress, USA; dissolved in complete medium) were co-incubated with 4 mmol/L L-kynurenine for 24 h, respectively.

Techniques: Activation Assay, Control, Immunostaining, Fluorescence, Expressing, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Type I gamma phosphatidylinositol phosphate kinase i5 suppresses YAP1 signaling

doi: 10.1016/j.jbc.2025.110573

Figure Lengend Snippet: PIPKIγi5 interacts with YAP1. A , HEK-293 cells coexpressing HA-PIPKIγi5 and FLAG-YAP1 were subjected to HA antibody immunoprecipitation and subsequently immunoblotted with the indicated antibodies. B , CAL27 cells were immunoprecipitated utilizing YAP1 antibody, then followed by immunoblotting with the indicated antibodies. C , recombinant FLAG-YAP1 and HA-PIPKIγi5 proteins (purified via Halo-tag system) were subjected into in vitro pull-down assays with Anti-FLAG Magnetic Beads. D , the diagram depicts the domain architecture of PIPKIγi1, i2, and i5. Developed with BioRender.com . E , the Myc-tagged PIPKIγi1, PIPKIγi2, PIPKIγi5, or PIPKIγi5 kinase dead mutants (D316A; referred to as PIPKIγi5KD) were coexpressed with FLAG-YAP1 in HEK-293 cells, followed by immunoprecipitation from cell lysates using anti-Myc antibody. F , quantification of YAP1 interaction with PIPKIγi1, PIPKIγi2, PIPKIγi5, or PIPKIγi5KD. G , the interaction between purified HA-PIPKIγi5 and FLAG-YAP1 was assessed using an in vitro solid-phase binding assay, with or without the presence of PI3,5P 2 or PI4,5P 2 as indicated. H , quantification of the interaction between PIPKIγi5 and YAP1 in the solid-phase binding assay. The values presented in the graphs indicate the mean ± SD from three independent experiments. Statistical significance was determined using one-way ANOVA and Tukey’s HSD ( F and H ) (∗∗∗ p < 0.0005; ∗∗∗∗ p < 0.0001). HEK-293, human embryonic kidney 293 cell line; HSD, honestly significant difference test; PIPKIγi5, type I gamma phosphatidylinositol phosphate kinase i5; YAP1, Yes-associated protein 1.

Article Snippet: YAP1 human recombinant protein (catalog no.: TP325864) was purchased from OriGene.

Techniques: Immunoprecipitation, Western Blot, Recombinant, Purification, In Vitro, Magnetic Beads, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Type I gamma phosphatidylinositol phosphate kinase i5 suppresses YAP1 signaling

doi: 10.1016/j.jbc.2025.110573

Figure Lengend Snippet: Identification of the binding regions responsible for the interaction between PIPKIγi5 and YAP1. A , a diagrammatic representation of the sequence of PIPKIγi5 C-terminal truncation mutants. Schematic diagram was created using BioRender.com . B , FLAG-YAP1 was coexpressed with HA-tagged wildtype PIPKIγi5 or a variety of PIPKIγi5 C-terminal truncation mutants, HA antibody was used for immunoprecipitation from cell lysates. C , levels of YAP1 interaction with wildtype PIPKIγi5 or PIPKIγi5 C-terminal truncation mutants were quantified. D , schematic representation of the domains of YAP1 and its truncation mutants. Schematic diagram was created using BioRender.com . E , MYC-PIPKIγi5 was coexpressed with HA-YAP1-N (1-263) or HA-YAP1-C (264-504), and MYC antibody was used for immunoprecipitation from cell lysates. The values presented in the graphs indicate the mean ± SD derived from three independent experiments. One-way ANOVA and Tukey’s HSD ( C ) (∗∗∗∗ p < 0.0001, and ns, nonsignificant). C, C terminus; HSD, honestly significant difference test; KD, kinase domain; N, N terminus; PDZ, PDZ-binding motif; PIPKIγi5, type I gamma phosphatidylinositol phosphate kinase i5; P-rich, proline-rich; SH3, SH3 domain; TAD, transcription activation domain; TEAD, transcriptional enhanced associate domain; WW, tryptophan–tryptophan domain; YAP1, Yes-associated protein 1.

Article Snippet: YAP1 human recombinant protein (catalog no.: TP325864) was purchased from OriGene.

Techniques: Binding Assay, Sequencing, Immunoprecipitation, Derivative Assay, Activation Assay

Journal: The Journal of Biological Chemistry

Article Title: Type I gamma phosphatidylinositol phosphate kinase i5 suppresses YAP1 signaling

doi: 10.1016/j.jbc.2025.110573

Figure Lengend Snippet: Loss of PIPKIγi5 enhances YAP1 target gene expression. Control siRNA or PIPKIγi5 siRNA-1 were transfected into CAL27 cells in low cell density ( e.g. , sparsity) ( A ) or high cell density ( e.g. , confluence) ( B ) conditions. The PIPKIγi5 protein levels were examined by Western blot in sparsity ( C ) and confluence cells ( D ). The mRNA levels of indicated YAP1 target genes were examined by real-time PCR ( E ). The values shown on graphs represent the mean ± SD from three independent experiments. One-way ANOVA and Tukey’s HSD ( E ) (∗ p < 0.05; ∗∗ p < 0.001; and ∗∗∗∗ p < 0.0001). HSD, honestly significant difference test; PIPKIγi5, type I gamma phosphatidylinositol phosphate kinase i5; YAP1, Yes-associated protein 1.

Article Snippet: YAP1 human recombinant protein (catalog no.: TP325864) was purchased from OriGene.

Techniques: Targeted Gene Expression, Control, Transfection, Western Blot, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Type I gamma phosphatidylinositol phosphate kinase i5 suppresses YAP1 signaling

doi: 10.1016/j.jbc.2025.110573

Figure Lengend Snippet: Effects of PIPKIγi5 on YAP1 expression and phosphorylation. CAL27 cells were transfected with either control, PIPKIγi5 siRNA-1, or PIPKIγi2 siRNA, and the specified protein levels were analyzed using Western blot ( A ). Quantification of total YAP1, YAP1 phosphorylation (S127 and S397), LATS1 and LATS2 in CAL27 cells ( B ). UM-SCC-1 cells were transfected with either control, PIPKIγi5 siRNA-1, or PIPKIγi2 siRNA, and the specified protein levels were analyzed using Western blot ( C ). Quantification of total YAP1, YAP1 phosphorylation (S127 and S397), LATS1 and LATS2 in UM-SCC-1 cells ( D ). The values shown on graphs represent the mean ± SD from three independent experiments. One-way ANOVA and Tukey’s HSD ( B , D ) (∗ p < 0.05, and ns, nonsignificant). HSD, honestly significant difference test; LATS1/2, large tumor suppressor kinase 1 and 2; PIPKIγi5, type I gamma phosphatidylinositol phosphate kinase i5; YAP1, Yes-associated protein 1.

Article Snippet: YAP1 human recombinant protein (catalog no.: TP325864) was purchased from OriGene.

Techniques: Expressing, Phospho-proteomics, Transfection, Control, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Type I gamma phosphatidylinositol phosphate kinase i5 suppresses YAP1 signaling

doi: 10.1016/j.jbc.2025.110573

Figure Lengend Snippet: PIPKIγi5 promotes YAP1 interaction with 14-3-3. A , FLAG-YAP1 were coexpressed with Myc-PIPKIγi5 or Myc-PIPKIγi5 KD in HEK-293 cells, and the cells were subjected to FLAG antibody immunoprecipitation and subsequently immunoblotted with the specified antibodies. B , quantification of YAP1 interaction with 14-3-3. C , HA-PIPKIγi5 was coexpressed with or without FLAG-YAP1 in HEK-293 cells, and then cells were subjected to HA antibody immunoprecipitation and subsequently immunoblotted with the specified antibodies. D , quantification of PIPKIγi5 interaction with 14-3-3. E , FLAG-YAP1 was expressed in control or PIPKIγi5-knockdown CAL27 cells and subjected to FLAG antibody immunoprecipitation and subsequently immunoblotted with the specified antibodies. F , quantification of YAP1 interaction with 14-3-3 in control or PIPKIγi5-knockdown cells. The values shown on graphs represent the mean ± SD from three independent experiments. One-way ANOVA and Tukey’s HSD ( B ) (∗ p < 0.05; ∗∗∗ p < 0.0005). Unpaired two-tailed Student’s t test ( D , F ) (∗ p < 0.05; ∗∗ p < 0.001). HEK-293, human embryonic kidney 293 cell line; HSD, honestly significant difference test; KD, kinase domain; PIPKIγi5, type I gamma phosphatidylinositol phosphate kinase i5; YAP1, Yes-associated protein 1.

Article Snippet: YAP1 human recombinant protein (catalog no.: TP325864) was purchased from OriGene.

Techniques: Immunoprecipitation, Control, Knockdown, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Type I gamma phosphatidylinositol phosphate kinase i5 suppresses YAP1 signaling

doi: 10.1016/j.jbc.2025.110573

Figure Lengend Snippet: Depletion of PIPKIγi5 increases the nuclear translocation of YAP1. CAL27 cells were transfected with either control siRNA or PIPKIγi5 siRNA-1. A , control or PIPKIγi5-knockdown CAL27 cells were stained with YAP1 antibody. Nuclei were stained with DAPI (scale bars represent 10 μm). High magnifications of the respective framed regions were shown on the right (scale bars represent 5 μm). B , quantification of YAP1 nuclear IF staining. Error bars indicate mean ± SD. (n = 60 cells from three independent experiments). Control or PIPKIγi5-knockdown CAL27 cells were treated with or without the MST1/2 inhibitor XMU-MP-1, and then the cytoplasmic and nuclear fractions were isolated using nuclear extraction kits. Cytoplasmic constituents were subjected to immunoblotting using indicated antibodies ( C ). Nuclear components were subjected to immunoblotting using specified antibodies ( D ). The levels of nuclear YAP1 in control or PIPKIγi5-knockdown CAL27 cells were quantified ( E ). The PIPKIγi5 expression levels, MST1/2 expression levels, and MST1/2 phosphorylation (MST1 (Thr183)/MST2 (Thr180)) levels were examined by Western blot ( F ). The values shown on graphs represent the mean ± SD from three independent experiments. Unpaired two-tailed Student’s t test ( B ) (∗∗∗ p < 0.0005). One-way ANOVA and Tukey’s HSD ( E ) (∗∗ p < 0.001; ∗∗∗ p < 0.0005, ∗∗∗∗ p < 0.0001). DAPI, 4′,6-diamidino-2-phenylindole; HSD, honestly significant difference test; IF, immunofluorescence; MST1/2, mammalian STE20-like protein kinase 1/2; PIPKIγi5, type I gamma phosphatidylinositol phosphate kinase i5; YAP1, Yes-associated protein 1

Article Snippet: YAP1 human recombinant protein (catalog no.: TP325864) was purchased from OriGene.

Techniques: Translocation Assay, Transfection, Control, Knockdown, Staining, Isolation, Extraction, Western Blot, Expressing, Phospho-proteomics, Two Tailed Test, Immunofluorescence

Journal: The Journal of Biological Chemistry

Article Title: Type I gamma phosphatidylinositol phosphate kinase i5 suppresses YAP1 signaling

doi: 10.1016/j.jbc.2025.110573

Figure Lengend Snippet: The kinase activity of PIPKIγi5 is required for the regulation of YAP1. A , the interaction between purified GST-14-3-3 and FLAG-YAP1 was assessed using an in vitro solid-phase binding assay, with or without the presence of PI3,5P 2 or PI4,5P 2 as specified. B , quantification of the interaction between 14-3-3 and YAP1 in the solid-phase binding assay. Myc-PIPKIγi5, Myc-PIPKIγi5KD, or Myc-PIPKIγi1 were individually expressed in either control or PIPKIγi5-knockdown CAL27 cells, and then PIPKIγi5 expression was identified using Western blot analysis ( C ), the expression of YAP1 target genes, ANKRD1 and CTGF, was assessed using real-time PCR ( D ). The values shown on graphs represent the mean ± SD from three independent experiments. One-way ANOVA and Tukey’s HSD ( B , D ) (∗∗ p < 0.001; ∗∗∗∗ p < 0.0001; and ns, nonsignificant). HSD, honestly significant difference test; PI3,5P 2, phosphatidylinositol-3,5-bisphosphate; PI4,5P 2 , phosphatidylinositol-4,5-bisphosphate; PIPKIγi5, type I gamma phosphatidylinositol phosphate kinase i5; YAP1, Yes-associated protein 1.

Article Snippet: YAP1 human recombinant protein (catalog no.: TP325864) was purchased from OriGene.

Techniques: Activity Assay, Purification, In Vitro, Binding Assay, Control, Knockdown, Expressing, Western Blot, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Type I gamma phosphatidylinositol phosphate kinase i5 suppresses YAP1 signaling

doi: 10.1016/j.jbc.2025.110573

Figure Lengend Snippet: Model for PIPKIγi5 regulation of Hippo/YAP1 signaling pathway. PIPKIγi5 interacts with YAP1, inhibiting its nuclear translocation and thereby suppressing YAP1-mediated gene transcription. By facilitating the interaction between YAP1 and 14-3-3 protein, PIPKIγi5 sequesters YAP1 in the cytosol. Therefore, reduction of PIPKIγi5 enhances YAP1 signaling. Schematic diagram was created using BioRender.com . PIPKIγi5, type I gamma phosphatidylinositol phosphate kinase i5; YAP1, Yes-associated protein 1.

Article Snippet: YAP1 human recombinant protein (catalog no.: TP325864) was purchased from OriGene.

Techniques: Translocation Assay

Journal: Nature Communications

Article Title: SKP2- and OTUD1-regulated non-proteolytic ubiquitination of YAP promotes YAP nuclear localization and activity

doi: 10.1038/s41467-018-04620-y

Figure Lengend Snippet: SKP2 interacts with YAP and induces its K63-linked polyubiquitination. a The HEK293T SFB-YAP stable cell line was transfected with MYC-tagged E3 ligases, followed by immunoprecipitation with anti-MYC beads and immunoblotting with antibodies against FLAG and MYC. b The HEK293T SFB-YAP stable cell line was co-transfected with the K63-specific mutant of HA-ubiquitin and the indicated E3 ligase, followed by pulldown with S-protein beads and immunoblotting with antibodies against HA and FLAG. c Co-immunoprecipitation of endogenous SKP2 with endogenous YAP. d In vitro binding of bacterially purified MBP-SKP2 to in vitro translated and purified GST-YAP. e In vitro binding of bacterially purified MBP-SKP2 to SFB-YAP purified from the HEK293T SFB-YAP stable cell line. MBP-SKP2 or MBP protein was incubated with SFB-YAP protein with or without CIP treatment at 37 °C for 1 h, followed by immunoblotting with antibodies against SKP2, p-YAP (S127), and FLAG. f The HEK293T SFB-YAP stable cell line was transfected with HA-ubiquitin (wild-type, K48R or K63R) and MYC-SKP2, followed by pulldown with S-protein beads and immunoblotting with antibodies against HA and FLAG. g The HEK293T SFB-YAP stable cell line was transfected with HA-ubiquitin (wild-type, K48-specific or K63-specific) and MYC-SKP2, followed by pulldown with S-protein beads and immunoblotting with antibodies against HA and FLAG. h Total K63-linkage specific ubiquitinated proteins in control or SKP2 knockout HEK293A cells (generated by CRISPR-Cas9) were immunoprecipitated by a K63-linked polyubiquitin-specific antibody, followed by immunoblotting with antibodies against YAP and ubiquitin. i siRNA targeting CUL1 or SKP1 was transfected into the HEK293T SFB-YAP stable cell line. Forty-eight hours after siRNA transfection, cells were transfected with HA-ubiquitin and MYC-SKP2, followed by pulldown with S-protein beads and immunoblotting with antibodies against HA and FLAG. j The HEK293T UBCH5c shRNA stable cell line was transfected with SFB-YAP and HA-ubiquitin with or without MYC-SKP2, followed by pulldown with S-protein beads and immunoblotting with antibodies against HA and FLAG. k Purified GST-YAP protein was incubated with ATP, E1, UBCH5c, and the K63-specific mutant of His-ubiquitin with or without the SCF SKP2 complex, followed by immunoprecipitation with a YAP-specific antibody and immunoblotting with antibodies against ubiquitin and YAP

Article Snippet: For pulldown of SFB-tagged proteins, cell extracts were incubated with S-protein beads (Millipore, 69704) at 4 °C for 2 h. For in vitro binding assays, purified GST-YAP (Novus Biologicals, H00010413-P01) was incubated with purified MBP-SKP2, and purified His-YAP (MyBioSource, MBS717875) was incubated with purified GST-OTUD1, followed by pulldown with Glutathione Sepharose beads (GE healthcare, 17-0756-01).

Techniques: Stable Transfection, Transfection, Immunoprecipitation, Western Blot, Mutagenesis, In Vitro, Binding Assay, Purification, Incubation, Knock-Out, Generated, CRISPR, shRNA

Journal: Nature Communications

Article Title: SKP2- and OTUD1-regulated non-proteolytic ubiquitination of YAP promotes YAP nuclear localization and activity

doi: 10.1038/s41467-018-04620-y

Figure Lengend Snippet: OTUD1 interacts with YAP and antagonizes its K63-linked ubiquitination. a SFB-tagged DUBs were co-transfected with MYC-YAP into HEK293T cells, followed by pulldown with S-protein beads and immunoblotting with antibodies against FLAG and MYC. b Seven SFB-DUBs were co-transfected with MYC-YAP and HA-ubiquitin into HEK293T cells, followed by immunoprecipitation with anti-MYC beads and immunoblotting with antibodies against HA and MYC. c The HEK293T SFB-YAP stable cell line was transfected with four YAP-interacting DUBs along with an 8× GTIIC luciferase reporter and a TK-Renilla luciferase reporter. Reporter activity was measured 48 h after transfection. Error bars are s.e.m. Statistical significance was determined by a two-tailed, unpaired Student’s t -test. * P < 0.05; ** P < 0.01; *** P < 0.001. n = 3 biological replicates. d Co-immunoprecipitation of endogenous OTUD1 with endogenous YAP. e In vitro binding of purified GST-OTUD1 to purified His-YAP. f HEK293T cells were co-transfected with SFB-OTUD1 (wild-type, C320S or H431R), HA-ubiquitin and MYC-YAP, followed by immunoprecipitation with anti-MYC beads and immunoblotting with antibodies against HA and MYC. g Ubiquitinated SFB-YAP was purified with S-protein beads and incubated with His-OTUD1 (wild-type or C320S) purified from bacteria. After the in vitro deubiquitination reaction, the bound proteins were eluted by boiling in Laemmli sample buffer and immunoblotted with antibodies against HA, FLAG, and OTUD1. h HEK293T cells were co-transfected with SFB-OTUD1, HA-ubiquitin (wild-type, K48R or K63R) and MYC-YAP, followed by immunoprecipitation with anti-MYC beads and immunoblotting with antibodies against HA and MYC. i siRNA targeting OTUD1 was transfected into the HEK293T SFB-YAP stable cell line. Forty-eight hours later, cells were transfected with HA-ubiquitin (wild-type, K48R or K63R), followed by pulldown with S-protein beads and immunoblotting with antibodies against HA and FLAG. j Total K63-linkage specific ubiquitinated proteins in control or OTUD1 knockout HEK293A cells (generated by CRISPR-Cas9) were immunoprecipitated by a K63-linked polyubiquitin-specific antibody, followed by immunoblotting with antibodies against YAP and ubiquitin

Article Snippet: For pulldown of SFB-tagged proteins, cell extracts were incubated with S-protein beads (Millipore, 69704) at 4 °C for 2 h. For in vitro binding assays, purified GST-YAP (Novus Biologicals, H00010413-P01) was incubated with purified MBP-SKP2, and purified His-YAP (MyBioSource, MBS717875) was incubated with purified GST-OTUD1, followed by pulldown with Glutathione Sepharose beads (GE healthcare, 17-0756-01).

Techniques: Transfection, Western Blot, Immunoprecipitation, Stable Transfection, Luciferase, Activity Assay, Two Tailed Test, In Vitro, Binding Assay, Purification, Incubation, Bacteria, Knock-Out, Generated, CRISPR

Journal: Cancers

Article Title: CARMA3 Promotes Colorectal Cancer Cell Motility and Cancer Stemness via YAP-Mediated NF-κB Activation

doi: 10.3390/cancers13235946

Figure Lengend Snippet: CARMA3/YAP/NF-κB/Slug signaling pathway is associated with a poor prognosis for human colorectal cancer. ( A ) Kaplan–Meier plot of overall survival of colorectal cancer patients with high and low YAP1 expression ( p < 0.05, log-rank test, HR = 1.82, Dataset: GSE17536). ( B , C ) Kaplan–Meier plot of disease-free survival of colorectal cancer patients with high and low RELA ( p < 0.05, log-rank test, HR = 12.85) or SNAI2 expression ( p < 0.05, log-rank test, HR = 1.71, Dataset: GSE17536). ( D , E ) The correlation between CARD10 and YAP1 expression or YAP1 and RELA expression in intestine cancer patients from the Correlation AnalyzeR database. Statistics from individual studies were obtained from the Correlation AnalyzeR database. The genes correlation (Pearson’s R) and p values were shown within each box plot. ( F ) The correlation between YAP1 and SNAI2 expression in colon cancer patients was analyzed from Oncomine datasets: Bittner_Colon. Statistics from individual studies were obtained from the Oncomine cancer database. The correlation coefficient ( r ), sample number ( N ), and p values were shown within the box plot. ( G ) A schematic model illustrating CARMA3/YAP/NF-κB/Slug pathway in CRC cells was shown. CARAM3 induces expression of YAP to activate NF-κB and induction of Slug, resulting in the enhancement of EMT, cancer stemness, and metastasis in CRC.

Article Snippet: Plasmids with YAP1 (cat. no. RC231269) and SLUG (cat. no. RC202365L1) expression were purchased from Origene (Rockville, MD, USA).

Techniques: Expressing

Journal: Cancer Research

Article Title: miR-375 Is Activated by ASH1 and Inhibits YAP1 in a Lineage-Dependent Manner in Lung Cancer

doi: 10.1158/0008-5472.can-11-1020

Figure Lengend Snippet: Figure 4. YAP1 inhibition by miR-375. A, histogram of gene expressions at 4 time points after Pre-miR-375 transfection. Gene expressions at 12 to 96 hours after transfection are shown as log2 ratio values between A549 cells transfected with Pre-miR-375 and Pre-miR-NC#2. Top, miR-375 predicted target genes; bottom, genes residing in chromosome 1, which was used as a control. B, time course of expression of each miR-375 predicted target gene after Pre-miR-375 transfection. C, Western blotting analysis of YAP1 protein. A549 cells transfected with Pre-miR-RNAs (15 nmol/L) were analyzed by Western blotting with the antibody against endogenous YAP1 protein. The intensity of YAP1 bands was determined with a densitometer and normalized with lamin B bands. Pre-miR-375 transfection downregulated the level of YAP1 protein by 58%. This downregulation is shown with a bar graph, in which the extent of downregulation is also numerically indicated. D, YAP1 30-UTR reporter assay. YAP1 mRNA contains 2 miR-375 target sites at the 30-UTR. Two reporter constructs, miR-375 1 and miR-375 2, carry 1 and 2 miR-375 sites, respectively. A549 cells were transfected with reporter constructs and Pre-miR-RNAs (10 nmol/L). The ratio of luciferase activity of Pre-miR-375 transfectants to that of Pre-miR-NC#2 transfectants is shown. miR-375 significantly suppressed luciferase activity in a target site-dependent manner. The alignment of 2 miR-375 target sites with mature miR-375 is also shown. The nucleotide positions in 30-UTR are indicated. E, negative correlations of miR-375 and YAP1 expressions in lung cancer cell lines. Left, YAP1 and miR-375 expressions showed significant negative correlations among the tested lung cancer cell lines. Right, YAP1 was frequently overexpressed in AD and SQ cells, whereas its expression was strongly suppressed in SCLC. In contrast, most SCLC showed overexpression of miR-375, whereas miR-375 was scarcely expressed in the AD and SQ cell lines. F, correlations of ASH1, miR-375, and YAP1 expression levels in primary lung cancer specimens. ASH1 and miR-375 expressions were positively correlated, whereas inverse correlations were present between miR-375 and YAP1, as well as between ASH1 and YAP1. NL, normal lung; AD, adenocarcinoma; SQ, squamous cell carcinoma; LA, large cell carcinoma; AS, adenosquamous carcinoma.

Article Snippet: Yes-associated protein 1 (YAP1) cDNA was purchased from OriGene and inserted into CSIIEF-MCS-IRES2-Venus.

Techniques: Inhibition, Transfection, Control, Expressing, Western Blot, Reporter Assay, Construct, Luciferase, Activity Assay, Over Expression

Journal: Cancer Research

Article Title: miR-375 Is Activated by ASH1 and Inhibits YAP1 in a Lineage-Dependent Manner in Lung Cancer

doi: 10.1158/0008-5472.can-11-1020

Figure Lengend Snippet: Figure 5. Lineage-dependent growth-regulating effects of YAP1 in lung cancers. A, fluorescent image of lentivirus-infected A549 and ACC-LC- 172 obtained 9 days after infection. Cells were infected with a YAP1- expressing or empty lentivirus carrying the fluorescent protein Venus. YAP1 and empty virus infection resulted in fluorescent signals in A549 cells. In contrast, fluorescence-positive cells were scarcely detected in ACC-LC-172 cells infected with the YAP1-virus, whereas noninfected cells grew well. B, flow cytometric analysis of fluorescence-positive cells. Lentivirus-infected cells were analyzed at 3 and 9 days after infection. The fluorescence-positive population was moderately increased in the A549 cells infected with the YAP1-virus, whereas that population almost disappeared in ACC-LC-172 cells infected with the YAP1-virus. C, Western blotting analysis of YAP1. A549 abundantly expressed endogenous and exogenous (HA-tagged) YAP1. In contrast, ACC-LC-172 did not express endogenous YAP1. In addition, exogenous YAP1 was scarcely detected. D, Western blotting analysis of caspases. Five days after infection, activated cleavage of caspase-7 and caspase-3 was detected in YAP1 virus-infected ACC-LC-172 cells. Normalized intensities of cleaved caspases are numerically indicated.

Article Snippet: Yes-associated protein 1 (YAP1) cDNA was purchased from OriGene and inserted into CSIIEF-MCS-IRES2-Venus.

Techniques: Infection, Expressing, Virus, Fluorescence, Western Blot

Journal: Carcinogenesis

Article Title: YAP1 is involved in mesothelioma development and negatively regulated by Merlin through phosphorylation.

doi: 10.1093/carcin/bgn200

Figure Lengend Snippet: Fig. 1. Mapping of amplified region of 11q22 locus in two MPM cases. (A) Details of array-CGH results of 11q22 amplification in two MPM cases. The signal ratios from array CGH analyses of two primary MPM cases (KD1033 and KD 1041) were plotted for all BAC clones based on chromosome position, and the results indicated discrete and significant amplifications at the 11q22 region. Amplifications in both cases were similar within a 3-Mb-long region, which was bounded by RP11-40B14 and RP11-652L13, and included only two BAC probes (RP11-203C2 and RP11-864G5), represented by open squares and closed diamonds, respectively. (B) Copy number analyses using quantitative PCR with TaqMan probes. Genes and registered transcripts within the 3-Mb-long region are shown. To further determine the boundaries of the amplified regions, the copy numbers of seven genes (PGR, TRPC6, ANGPTL5, YAP1, BIRC2, MMP13 and AB08258, indicated by boxes) were investigated using TaqMan probes. Four genes (ANGPTL5, YAP1, BIRC2 and MMP13) showed high copy numbers in the two MPM cases, suggesting that both carried quite similar 1-Mb-long amplifications.

Article Snippet: YAP1 cDNA was purchased from OriGene (Rockville, MD) and inserted into pcDNA (Invitrogen, Carlsbad, CA) and pEGFP-C1 (Clontech, Mountain View, CA) vectors.

Techniques: Clone Assay, Real-time Polymerase Chain Reaction

Journal: Carcinogenesis

Article Title: YAP1 is involved in mesothelioma development and negatively regulated by Merlin through phosphorylation.

doi: 10.1093/carcin/bgn200

Figure Lengend Snippet: Fig. 2. Alterations of copy number and expression of YAP1 and BIRC2. (A) Expression analyses of the ANGPTL5, YAP1, BIRC2 and BIRC3 genes indicated that YAP1 and BIRC2 were overexpressed in common in both MPM cases with amplification. (B) Immunohistochemical staining of YAP1. Immunohistochemical analysis clearly demonstrated overexpression and nuclear accumulation of YAP1 in KD1033 (left panel), whereas normal pleural mesothelial cells did not show any YAP1 signals (arrowheads, right panel). (C) Expression analysis in primary MPM cases. In an examination of 14 primary MPM cases and the normal mesothelial cell line MeT-5A, the two MPM cases showed the greatest amount of upregulated expressions of the YAP1 and BIRC2 genes with amplification. (D) Copy number analysis of primary MPM cases. In an examination of 14 MPM cases and the normal mesothelial cell line MeT-5A, only the two MPM cases demonstrated amplification of the YAP1 and BIRC2 genes.

Article Snippet: YAP1 cDNA was purchased from OriGene (Rockville, MD) and inserted into pcDNA (Invitrogen, Carlsbad, CA) and pEGFP-C1 (Clontech, Mountain View, CA) vectors.

Techniques: Expressing, Immunohistochemical staining, Staining, Over Expression

Journal: Carcinogenesis

Article Title: YAP1 is involved in mesothelioma development and negatively regulated by Merlin through phosphorylation.

doi: 10.1093/carcin/bgn200

Figure Lengend Snippet: Fig. 3. Involvement of YAP1 in cell proliferation. (A) Knockdown of endogenous YAP1 in MPM cell line NCI-H290. Both YAP1-RNAi vectors, YAP1-sh1 and YAP1-sh2, showed effective suppression of the level of YAP1 protein, whereas the control vectors, GFP-sh and YAP1-scr1, showed no inhibition. (B) Inhibition of NCI-H290 proliferation by YAP1-RNAi. Colorimetric assay results demonstrated that both the YAP1-sh1 and YAP1-sh2 vectors induced significant suppression of cell proliferation. YAP1-sh2 inhibited proliferation to a greater degree as compared with YAP1-sh1, consistent with the RNAi effect. (C) Cell-cycle arrest by YAP1-RNAi. YAP1-sh2 clearly induced G1 arrest of NCI-H290 cells. (D) Induction of sub-G1 population by YAP1-RNAi. The sub-G1 population of NCI-H290 cells was induced by both YAP1-RNAi vectors, though induction by YAP1-sh2 was greater. (E) Promotion of MeT-5A cell proliferation by YAP1 overexpression. Although YAP1 overexpression did not show a clear effect in the usual condition [fetal calf serum (FCS) 5%], YAP1 overexpression moderately promoted cell proliferation in the low-serum condition (FCS 1%).

Article Snippet: YAP1 cDNA was purchased from OriGene (Rockville, MD) and inserted into pcDNA (Invitrogen, Carlsbad, CA) and pEGFP-C1 (Clontech, Mountain View, CA) vectors.

Techniques: Knockdown, Control, Inhibition, Colorimetric Assay, Over Expression

Journal: Carcinogenesis

Article Title: YAP1 is involved in mesothelioma development and negatively regulated by Merlin through phosphorylation.

doi: 10.1093/carcin/bgn200

Figure Lengend Snippet: Fig. 4. Functional interaction between YAP1 and Merlin. (A and B) Induction of YAP1 phosphorylation by Merlin. (A) NF2-null NCI-H290 cells were transfected with YAP1 and NF2 expression vectors. Cotransfection of NF2 significantly induced phosphorylation of the YAP1 S 127 residue. (B) Either full-length or truncated NF2 was introduced into NCI-H290 cells with a lentivirus. Phosphorylation of YAP1 was induced by the full-length NF2 lentivirus in a dose-dependent manner, whereas the truncated NF2 FERM lentivirus did not have any clear effect. (C) Immunoblotting of cytoplasmic and nuclear fractions. NCI-H290 cells were transfected with wild-type (wt) or phosphorylation-defective S 127A mutant (mut) YAP1 and NF2 expression vectors. Immunoblots of nuclear and cytoplasmic fractions of transfectants clearly showed that phospho-YAP1 was mainly localized in the cytoplasm, as detected by the anti-S 127 phospho-YAP1 antibody, though total YAP1 was localized in both the nucleus and cytoplasm. In addition, the nuclear/cytoplasmic ratio of YAP1 and phospho-YAP1 proteins was measured with a densitometer and indicated with a bar graph, which clearly demonstrates the tight cytoplasmic retention of wild-type phospho-YAP1 protein. No signals were detected in mutant YAP1 transfectants with the anti- phospho-YAP1 antibody (asterisks). Results of immunoblotting with antibodies against a-tubulin and nuclear laminB1 indicated that proper fractionation occurred. (D) Reduction of YAP1 nuclear localization by NF2. NCI-H290 cells were transfected with expression vectors of the EGFP-fused wild-type (wt) or S 127A mutant (mut) YAP1, together with the vector control or V5-tagged NF2. Both the wild-type and mutant YAP1 proteins localize in both the nuclei and cytoplasm of H290 cells. Cotransfection of NF2 reduced nuclear localization of wild-type YAP1, whereas the localization of mutant YAP1 was not affected by NF2.

Article Snippet: YAP1 cDNA was purchased from OriGene (Rockville, MD) and inserted into pcDNA (Invitrogen, Carlsbad, CA) and pEGFP-C1 (Clontech, Mountain View, CA) vectors.

Techniques: Functional Assay, Phospho-proteomics, Transfection, Expressing, Cotransfection, Residue, Western Blot, Mutagenesis, Fractionation, Plasmid Preparation, Control

Journal: Carcinogenesis

Article Title: YAP1 is involved in mesothelioma development and negatively regulated by Merlin through phosphorylation.

doi: 10.1093/carcin/bgn200

Figure Lengend Snippet: Fig. 5. Physical interaction between YAP1 and Merlin. (A) Immunoprecipitation: 293T cells were transfected with V5-tagged NF2 and/ or YAP1 expression vectors, then Merlin was immunoprecipitated with the anti-V5 antibody and coprecipitation with YAP1 was studied. Both wild-type and S 127A mutant YAP1 were coprecipitated with Merlin. The anti- phospho-YAP1 antibody did not show any clear signals. (B) In vitro pull- down assay. Cell lysates of 293T cells were transfected with a YAP1 or NHERF1 vector, then incubated with GST-alone or GST—Merlin-bound beads. Immunoblots of bead-bound proteins (Bound) and the initial cell lysates (Input) are shown. Both YAP1 and NHERF1 proteins bound to GST– Merlin, though NHERF1 did not enhance the association of YAP1 with GST–Merlin. In the immunoblot of bound NHERF1, non-specific bands are indicated by asterisks.

Article Snippet: YAP1 cDNA was purchased from OriGene (Rockville, MD) and inserted into pcDNA (Invitrogen, Carlsbad, CA) and pEGFP-C1 (Clontech, Mountain View, CA) vectors.

Techniques: Immunoprecipitation, Transfection, Expressing, Mutagenesis, In Vitro, Pull Down Assay, Plasmid Preparation, Incubation, Western Blot

Journal: Neurology International

Article Title: YAP1 Upregulates Cytoskeleton Regulator ARHGEF1 and Tissue Regeneration Factor NEDD9 in a Multiplex Proteomic Study

doi: 10.3390/neurolint18050096

Figure Lengend Snippet: ( A )YAP1 protein is expressed in different cell types relevant to the brain, such as astrocytes (CCF-STTG1), microglia (HMC3), neuroblastoma (SH-SY5Y), and NT2 cells, as demonstrated by immunoblotting. Data are mean ± SEM ( n = 3). ( B ) Images show immunocytochemical evidence of YAP1 expression (in green) and DAPI-stained nuclei (in blue) in different cell types and human neurons. Scale: 20 µm.

Article Snippet: We cultured the immortalized human neuroblastoma cell line SH-SY5Y (cat # CRL2266, ATCC) in DMEM/F12 medium and infected only once with Lenti ORF particles, human YAP1 (mGFP-tagged) transcript variant 1 (cat # RC225864L4V, Origene, Rockville, MD, USA), and generated stable cells expressing YAP1-GFP with puromycin selection at 5.0 μg per milliliter for two weeks.

Techniques: Western Blot, Expressing, Staining

Journal: Neurology International

Article Title: YAP1 Upregulates Cytoskeleton Regulator ARHGEF1 and Tissue Regeneration Factor NEDD9 in a Multiplex Proteomic Study

doi: 10.3390/neurolint18050096

Figure Lengend Snippet: YAP1 protein levels are significantly downregulated in the nuclear fractions but not the cytosolic fractions of the AD brains relative to NC brains. Brain lysates were immunoblotted, and protein levels were quantified by normalizing to HDAC2 levels for nuclear fractions and to GAPDH levels for cytosolic fractions. Data are mean ± SEM ( n = 5 per group). *** p < 0.001 by t -test.

Article Snippet: We cultured the immortalized human neuroblastoma cell line SH-SY5Y (cat # CRL2266, ATCC) in DMEM/F12 medium and infected only once with Lenti ORF particles, human YAP1 (mGFP-tagged) transcript variant 1 (cat # RC225864L4V, Origene, Rockville, MD, USA), and generated stable cells expressing YAP1-GFP with puromycin selection at 5.0 μg per milliliter for two weeks.

Techniques:

Journal: Neurology International

Article Title: YAP1 Upregulates Cytoskeleton Regulator ARHGEF1 and Tissue Regeneration Factor NEDD9 in a Multiplex Proteomic Study

doi: 10.3390/neurolint18050096

Figure Lengend Snippet: Antibody-based multiplex microarray screening of 8000 proteins revealed alterations of specific proteins. ( A ) SH-SY5Y cells expressing YAP1-GFP. Scale, 50 µm. ( B ) YAP1-GFP expression supported by immunoblot. ( C ) Examples of glass slide luminescence. ( D ) Heatmap of control and YAP1 biomarkers in 8000 samples. The data were imported to JMP Genomics 10.2 for Hierarchical Cluster analysis.

Article Snippet: We cultured the immortalized human neuroblastoma cell line SH-SY5Y (cat # CRL2266, ATCC) in DMEM/F12 medium and infected only once with Lenti ORF particles, human YAP1 (mGFP-tagged) transcript variant 1 (cat # RC225864L4V, Origene, Rockville, MD, USA), and generated stable cells expressing YAP1-GFP with puromycin selection at 5.0 μg per milliliter for two weeks.

Techniques: Multiplex Assay, Microarray, Expressing, Western Blot, Control

Journal: Neurology International

Article Title: YAP1 Upregulates Cytoskeleton Regulator ARHGEF1 and Tissue Regeneration Factor NEDD9 in a Multiplex Proteomic Study

doi: 10.3390/neurolint18050096

Figure Lengend Snippet: YAP1 expression increased markers of synaptic endocytosis and ATP-dependent activity. ( A ) Gene ontology (GO) biological process over-representation analysis with 283 differentially expressed biomarkers between YAP1 and control samples. ( B ) GO cellular component over-representation analysis with 283 differentially expressed biomarkers between control and YAP1-expressing SH-SY5Y cells.

Article Snippet: We cultured the immortalized human neuroblastoma cell line SH-SY5Y (cat # CRL2266, ATCC) in DMEM/F12 medium and infected only once with Lenti ORF particles, human YAP1 (mGFP-tagged) transcript variant 1 (cat # RC225864L4V, Origene, Rockville, MD, USA), and generated stable cells expressing YAP1-GFP with puromycin selection at 5.0 μg per milliliter for two weeks.

Techniques: Expressing, Activity Assay, Control

Journal: Neurology International

Article Title: YAP1 Upregulates Cytoskeleton Regulator ARHGEF1 and Tissue Regeneration Factor NEDD9 in a Multiplex Proteomic Study

doi: 10.3390/neurolint18050096

Figure Lengend Snippet: ( A ) GO molecular function over-representation analysis with 283 differentially expressed biomarkers between YAP1 and control samples. ( B ) Interaction network of human YAP1 protein as analyzed by STRING.

Article Snippet: We cultured the immortalized human neuroblastoma cell line SH-SY5Y (cat # CRL2266, ATCC) in DMEM/F12 medium and infected only once with Lenti ORF particles, human YAP1 (mGFP-tagged) transcript variant 1 (cat # RC225864L4V, Origene, Rockville, MD, USA), and generated stable cells expressing YAP1-GFP with puromycin selection at 5.0 μg per milliliter for two weeks.

Techniques: Control

Journal: Neurology International

Article Title: YAP1 Upregulates Cytoskeleton Regulator ARHGEF1 and Tissue Regeneration Factor NEDD9 in a Multiplex Proteomic Study

doi: 10.3390/neurolint18050096

Figure Lengend Snippet: The bar graph shows GO analysis results of the increased ( A ) and decreased proteins ( B ) due to YAP1 expression using ShinyGO 0.85.1. Sets of nonredundant, significant GO enrichment terms are displayed to show the foldenrichment and the number of proteins matched to each term. The fold-enrichment values are displayed with the highest on top, and the shade of each bar reflects the number of proteins found in a given GO category.

Article Snippet: We cultured the immortalized human neuroblastoma cell line SH-SY5Y (cat # CRL2266, ATCC) in DMEM/F12 medium and infected only once with Lenti ORF particles, human YAP1 (mGFP-tagged) transcript variant 1 (cat # RC225864L4V, Origene, Rockville, MD, USA), and generated stable cells expressing YAP1-GFP with puromycin selection at 5.0 μg per milliliter for two weeks.

Techniques: Expressing

Journal: Neurology International

Article Title: YAP1 Upregulates Cytoskeleton Regulator ARHGEF1 and Tissue Regeneration Factor NEDD9 in a Multiplex Proteomic Study

doi: 10.3390/neurolint18050096

Figure Lengend Snippet: Evidence of increased levels of ( A ) NEDD9 and ( B ) ARHGEF1 inYAP1-GFP-expressing SH-SY5Y cells when compared to control cells by immunoblotting. ( C ) The LC3-16/LC3-18 ratio was also increased, indicating YAP1 activates autophagy. However, levels of ( D ) p16INK4a, a marker of senescence, were almost completely abolished. Data are mean + SEM, n = 3/group. *, p < 0.05, **, p < 0.01, ***, p < 0.001 by t -test.

Article Snippet: We cultured the immortalized human neuroblastoma cell line SH-SY5Y (cat # CRL2266, ATCC) in DMEM/F12 medium and infected only once with Lenti ORF particles, human YAP1 (mGFP-tagged) transcript variant 1 (cat # RC225864L4V, Origene, Rockville, MD, USA), and generated stable cells expressing YAP1-GFP with puromycin selection at 5.0 μg per milliliter for two weeks.

Techniques: Expressing, Control, Western Blot, Marker

Journal: PLoS ONE

Article Title: miR-7977 inhibits the Hippo-YAP signaling pathway in bone marrow mesenchymal stromal cells

doi: 10.1371/journal.pone.0213220

Figure Lengend Snippet: Summary of results of GSEA showing p-value <0.00001 and q-value <0.25.

Article Snippet: Expression plasmid of human miR-7977 (MIR7977-MiRNA) and empty vector control (pCMV-MIR) were purchased from OriGene Inc. MSCs or HTS-5 cells were transfected with 2 μg of the 8xGTIIC-luciferase, YAP1 (GFP-tagged)-pCMV6-AC-GFP, control pMirTarget plasmid (pCMV-MIR) or pCMV-MIR-7977 using Lipofectamine LTX transfection reagent (Life Technologies).

Techniques:

Journal: PLoS ONE

Article Title: miR-7977 inhibits the Hippo-YAP signaling pathway in bone marrow mesenchymal stromal cells

doi: 10.1371/journal.pone.0213220

Figure Lengend Snippet: (A) Enrichment plots from GSEA are shown. The panel was obtained by GSEA using the gene set database, c6all.v6.0.symbols.gmt [Oncogenic signature]. (B) The core enrichment of genes was visualized as heatmap. Left side showed control MSCs and Right side showed miR-7977-transduced MSCs. (C) The expression of each gene associated with YAP1 and tight junction was visualized as a molecular interaction network made by Cytoscape software. Gene symbols are overlaid on each node and interactions between genes are shown as dotted lines with an arrow (edge). The fold change of gene expression [Log 2 (miR-7977 transduced MSCs/ Control transduced MSCs)] was superimposed on the molecular interaction network.

Article Snippet: Expression plasmid of human miR-7977 (MIR7977-MiRNA) and empty vector control (pCMV-MIR) were purchased from OriGene Inc. MSCs or HTS-5 cells were transfected with 2 μg of the 8xGTIIC-luciferase, YAP1 (GFP-tagged)-pCMV6-AC-GFP, control pMirTarget plasmid (pCMV-MIR) or pCMV-MIR-7977 using Lipofectamine LTX transfection reagent (Life Technologies).

Techniques: Expressing, Software

Journal: PLoS ONE

Article Title: miR-7977 inhibits the Hippo-YAP signaling pathway in bone marrow mesenchymal stromal cells

doi: 10.1371/journal.pone.0213220

Figure Lengend Snippet: (A) qRT-PCR for STK4 was conducted after transduction of control or miR-7977 for primary MSCs (5 nM) and HTS-5 (20 nM). The left panel shows the results in MSC (n = 5), and the right panel shows the results in the HTS-5 mesenchymal cell line (n = 3). (B) Immunoblot analysis of Hippo signaling pathway after transfer of control siRNA or miR-7977 into HTS-5. Anti-STK4 Ab, anti-YAP1 and anti- phospho-YAP1 Ser127 were used. As internal standard, anti-β-actin was used.

Article Snippet: Expression plasmid of human miR-7977 (MIR7977-MiRNA) and empty vector control (pCMV-MIR) were purchased from OriGene Inc. MSCs or HTS-5 cells were transfected with 2 μg of the 8xGTIIC-luciferase, YAP1 (GFP-tagged)-pCMV6-AC-GFP, control pMirTarget plasmid (pCMV-MIR) or pCMV-MIR-7977 using Lipofectamine LTX transfection reagent (Life Technologies).

Techniques: Quantitative RT-PCR, Transduction, Western Blot

Journal: PLoS ONE

Article Title: miR-7977 inhibits the Hippo-YAP signaling pathway in bone marrow mesenchymal stromal cells

doi: 10.1371/journal.pone.0213220

Figure Lengend Snippet: (A) Nuclear localization of GFP-tagged YAP1 after transduction of YAP1 (GFP-tagged)—pCMV6-AC-GFP into HTS-5. Subsequently, control or 20 nM miR-7977 mimic was transfected, and the cells were visualized. Left panel: control siRNA transfer. Right panel: miR-7977 transfer. Scale = 10 μm. (B) Schematic diagram of luciferase assay to evaluate the interaction of TEAD and YAP1. (C) Transduction of pCMV-MIR-7977 and 8xGTIIC-luciferase into the HTS-5 stromal cell line and analysis of luciferase activity 2 days after transduction. Luciferase activity was normalized to the activity of an internal control (renilla luciferase). * P < 0.05, pCMV-MIR (control, n = 8) vs. pCMV-MIR-7977 (n = 8) (Student’s t -test). Data represent three independent experiments, each done in octuplicate.

Article Snippet: Expression plasmid of human miR-7977 (MIR7977-MiRNA) and empty vector control (pCMV-MIR) were purchased from OriGene Inc. MSCs or HTS-5 cells were transfected with 2 μg of the 8xGTIIC-luciferase, YAP1 (GFP-tagged)-pCMV6-AC-GFP, control pMirTarget plasmid (pCMV-MIR) or pCMV-MIR-7977 using Lipofectamine LTX transfection reagent (Life Technologies).

Techniques: Transduction, Transfection, Luciferase, Activity Assay