Journal: iScience

Article Title: Promotion of breast cancer by the DNPH1 enzyme

doi: 10.1016/j.isci.2026.115227

Figure Lengend Snippet: Alterations in mammary tumors upon Dnph1 knockout (A) Examples of CD31 staining in mammary tumors from a MMTV-Her2/Dnph1 +/+ and a MMTV-Her2/Dnph1 −/− mouse. Right panels show ImageJ-mediated image modulation to enhance vessel structures, allowing the computer-mediated quantification of blood vessel density. Scale bars, 0.4 mm. (B) Corresponding quantification of vessel density. Tumors were age- and weight-matched; two-tailed, unpaired t test. (C) Tumor vessel density plotted against the weight of the tumor. These tumors were not weight- or age-matched, yet include those shown in panel B. Shown are linear regressions; while the slopes were not significantly different, the Y-intercepts were significantly different ( p = 0.0007). (D) Relative levels of AMP, GMP, and UMP in mammary tumors from MMTV-Her2/Dnph1 +/+ and MMTV-Her2/Dnph1 −/− mice; two-tailed, unpaired t test. (E and F) Immunohistochemical staining for phospho-AMPKα or phospho-YAP1, respectively, on age- and weight-matched tumors as in panel B. Examples of immunohistochemical staining are shown at the bottom. Scale bars, 0.4 mm. Means with standard deviation are shown in panels B and D-F.

Article Snippet: YAP1 phosphorylated on serine 127 , Cell Signaling , Cat# 4911.

Techniques: Knock-Out, Staining, Two Tailed Test, Immunohistochemical staining, Standard Deviation

Journal: iScience

Article Title: Promotion of breast cancer by the DNPH1 enzyme

doi: 10.1016/j.isci.2026.115227

Figure Lengend Snippet: Model how DNPH1 promotes breast tumor development and progression This model is centered around a DNPH1-YAP1 axis, yet DNPH1 may utilize to-be-identified effectors other than YAP1 to stimulate, e.g., stem cells or angiogenesis.

Article Snippet: YAP1 phosphorylated on serine 127 , Cell Signaling , Cat# 4911.

Techniques:

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: Antibodies to YAP1 (catalog no.: 14074S), GST-tag (catalog no.: 2622S), HA-tag (catalog no.: 3724S), MYC-tag (catalog no.: 2276S), CTGF (catalog no.: 86641T), CYR61 (catalog no.: 14479T), LATS1 (catalog no.: 3477S), LATS2 (catalog no.:5888S), GAPDH (catalog no.: 5174S), phosphorylated YAP1 (Ser127, catalog no.: 13008T), and phosphorylated YAP1 (Ser397, catalog no.: 13619T), Lamin B1 (catalog no.: 12586S), MST1 (catalog no.: 14946), MST2 (catalog no.: 3952), and phosphorylated MST1/2 (catalog no.: 49332) were from Cell Signaling Technology.

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: Antibodies to YAP1 (catalog no.: 14074S), GST-tag (catalog no.: 2622S), HA-tag (catalog no.: 3724S), MYC-tag (catalog no.: 2276S), CTGF (catalog no.: 86641T), CYR61 (catalog no.: 14479T), LATS1 (catalog no.: 3477S), LATS2 (catalog no.:5888S), GAPDH (catalog no.: 5174S), phosphorylated YAP1 (Ser127, catalog no.: 13008T), and phosphorylated YAP1 (Ser397, catalog no.: 13619T), Lamin B1 (catalog no.: 12586S), MST1 (catalog no.: 14946), MST2 (catalog no.: 3952), and phosphorylated MST1/2 (catalog no.: 49332) were from Cell Signaling Technology.

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: Antibodies to YAP1 (catalog no.: 14074S), GST-tag (catalog no.: 2622S), HA-tag (catalog no.: 3724S), MYC-tag (catalog no.: 2276S), CTGF (catalog no.: 86641T), CYR61 (catalog no.: 14479T), LATS1 (catalog no.: 3477S), LATS2 (catalog no.:5888S), GAPDH (catalog no.: 5174S), phosphorylated YAP1 (Ser127, catalog no.: 13008T), and phosphorylated YAP1 (Ser397, catalog no.: 13619T), Lamin B1 (catalog no.: 12586S), MST1 (catalog no.: 14946), MST2 (catalog no.: 3952), and phosphorylated MST1/2 (catalog no.: 49332) were from Cell Signaling Technology.

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: Antibodies to YAP1 (catalog no.: 14074S), GST-tag (catalog no.: 2622S), HA-tag (catalog no.: 3724S), MYC-tag (catalog no.: 2276S), CTGF (catalog no.: 86641T), CYR61 (catalog no.: 14479T), LATS1 (catalog no.: 3477S), LATS2 (catalog no.:5888S), GAPDH (catalog no.: 5174S), phosphorylated YAP1 (Ser127, catalog no.: 13008T), and phosphorylated YAP1 (Ser397, catalog no.: 13619T), Lamin B1 (catalog no.: 12586S), MST1 (catalog no.: 14946), MST2 (catalog no.: 3952), and phosphorylated MST1/2 (catalog no.: 49332) were from Cell Signaling Technology.

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: Antibodies to YAP1 (catalog no.: 14074S), GST-tag (catalog no.: 2622S), HA-tag (catalog no.: 3724S), MYC-tag (catalog no.: 2276S), CTGF (catalog no.: 86641T), CYR61 (catalog no.: 14479T), LATS1 (catalog no.: 3477S), LATS2 (catalog no.:5888S), GAPDH (catalog no.: 5174S), phosphorylated YAP1 (Ser127, catalog no.: 13008T), and phosphorylated YAP1 (Ser397, catalog no.: 13619T), Lamin B1 (catalog no.: 12586S), MST1 (catalog no.: 14946), MST2 (catalog no.: 3952), and phosphorylated MST1/2 (catalog no.: 49332) were from Cell Signaling Technology.

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: Antibodies to YAP1 (catalog no.: 14074S), GST-tag (catalog no.: 2622S), HA-tag (catalog no.: 3724S), MYC-tag (catalog no.: 2276S), CTGF (catalog no.: 86641T), CYR61 (catalog no.: 14479T), LATS1 (catalog no.: 3477S), LATS2 (catalog no.:5888S), GAPDH (catalog no.: 5174S), phosphorylated YAP1 (Ser127, catalog no.: 13008T), and phosphorylated YAP1 (Ser397, catalog no.: 13619T), Lamin B1 (catalog no.: 12586S), MST1 (catalog no.: 14946), MST2 (catalog no.: 3952), and phosphorylated MST1/2 (catalog no.: 49332) were from Cell Signaling Technology.

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: Antibodies to YAP1 (catalog no.: 14074S), GST-tag (catalog no.: 2622S), HA-tag (catalog no.: 3724S), MYC-tag (catalog no.: 2276S), CTGF (catalog no.: 86641T), CYR61 (catalog no.: 14479T), LATS1 (catalog no.: 3477S), LATS2 (catalog no.:5888S), GAPDH (catalog no.: 5174S), phosphorylated YAP1 (Ser127, catalog no.: 13008T), and phosphorylated YAP1 (Ser397, catalog no.: 13619T), Lamin B1 (catalog no.: 12586S), MST1 (catalog no.: 14946), MST2 (catalog no.: 3952), and phosphorylated MST1/2 (catalog no.: 49332) were from Cell Signaling Technology.

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: Antibodies to YAP1 (catalog no.: 14074S), GST-tag (catalog no.: 2622S), HA-tag (catalog no.: 3724S), MYC-tag (catalog no.: 2276S), CTGF (catalog no.: 86641T), CYR61 (catalog no.: 14479T), LATS1 (catalog no.: 3477S), LATS2 (catalog no.:5888S), GAPDH (catalog no.: 5174S), phosphorylated YAP1 (Ser127, catalog no.: 13008T), and phosphorylated YAP1 (Ser397, catalog no.: 13619T), Lamin B1 (catalog no.: 12586S), MST1 (catalog no.: 14946), MST2 (catalog no.: 3952), and phosphorylated MST1/2 (catalog no.: 49332) were from Cell Signaling Technology.

Techniques: Translocation Assay

Journal: Science Advances

Article Title: Liver regeneration–associated hepatocellular YAP1 activation prevents colorectal cancer liver metastasis through glutamine competition

doi: 10.1126/sciadv.adw6926

Figure Lengend Snippet: ( A ) GSEA of RNA-seq data from mouse livers 3 days after hepatectomy. ( B ) Heatmap of gene expressions of YAP1 conserved signature in RNA-seq data from mouse liver in sham, 30% PHx, and 70% PHx groups 3 days after surgeries. ( C and D ) IHC and WB analyses of YAP1 S127 and nuclear YAP1 in the remaining livers. ( E ) qRT-PCR analysis of YAP1 downstream genes Ccn1 and Ccn2 levels in the remnant liver. ( F ) Schematic of the CRLM mouse model with/without YAP1 overexpression in hepatocytes generated via AAV8. ( G and H ) Liver–to–body weight ratios and tumor-to-liver volume ratios of the negative control (NC) and overexpression (OE) groups. ( I ) Schematic, images, IF, and hematoxylin and eosin (H&E) staining of livers from the NC or OE groups. ( J ) Survival curve of the CRLM model mice in the NC and OE groups. ( K ) Schematic of the CRLM mouse model with/without hepatocellular YAP1 overexpression and verteporfin administration. ( L and M ) Liver–to–body weight ratios and tumor-to-liver volume ratios of the mice in the NC, YAP1 OE, and YAP1 OE with verteporfin groups. ( N ) Schematic, images, MRI scans, and IF and H&E staining of livers and lungs from CRLM model mice in the above three groups. ( O ) Survival curve of the CRLM model mice in the above three groups. ( P and Q ) WB and IHC analyses of EGFR levels in the remnant liver tissue in sham, 30% PHx, and 70% PHx groups. ( R to T ) WB and IF analyses of YAP1 levels in AML12 cells after the addition of EGF and canertinib. (T) WB analysis of EGFR and YAP1 levels after major hepatectomy and the addition of canertinib. ( U ) CoIP analysis showing the combination of the YAP1 and EGFR proteins. IgG, immunoglobulin G.

Article Snippet: The primary antibodies used for IHC, IF, and WB analyses were as follows: YAP1 (14074, CST, USA), YAP1 S127 (13008, CST), CXCL5 (DF9919, Affinity, USA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (2118, CST), LATS1 (3477, CST), p-LATS1 (9157, CST), lamin B1 (12987-1-AP, Proteintech, USA), connective tissue growth factor (CTGF; ab227180, Abcam, UK), Cystein Rich 61 (Cyr61; ab228592, Abcam), GLS (12855-1-AP, Proteintech), GOT1 (34423, CST), GLUL (80636, CST), PSAT1 (ab154055, Abcam), SLC1A5 (ab237704, Abcam), SLC7A5 (32683, CST), and SLC38A1 (28632-1-AP, Proteintech).

Techniques: RNA Sequencing, Quantitative RT-PCR, Over Expression, Generated, Negative Control, Staining

Journal: Science Advances

Article Title: Liver regeneration–associated hepatocellular YAP1 activation prevents colorectal cancer liver metastasis through glutamine competition

doi: 10.1126/sciadv.adw6926

Figure Lengend Snippet: ( A ) Schematic of sorting MC38 cells from mouse CRLMs and a heatmap of the RNA-seq results of the sham and 70% PHx groups. ( B ) Left: Venn diagram presenting the overlapping chemokine-related genes and DEGs from RNA-seq. Right: List of the top 5 differentially expressed chemokine-related genes. ( C ) Representative images of the mouse cytokine array of the tumors of the CRLM model mice in the two groups. ( D ) Representative images of IHC staining for CXCL5 in the tumors of the CRLM model mice in sham, 30% PHx, and 70% PHx groups. ( E ) GSEA of the RNA-seq data on YAP1 signaling in sham and 70% PHx groups. ( F and G ) Representative images of IHC staining for YAP1 S127 and YAP1 in tumors of the CRLM model mice in sham, 30% PHx, and 70% PHx groups. ( H ) WB analysis of YAP1 levels in the nuclei of mouse CRLMs and adjacent livers in sham, 30% PHx, and 70% PHx groups. ( I ) ChIP analysis of the combination of the YAP1 protein and the Cxcl5 promoter via qPCR in triplicate. ( J and K ) Levels of the CXCL5 protein in the culture medium of MC38 cells with YAP1 overexpression (J) or knockdown (K). ( L and M ) Top left: Workflow for the Transwell migration assay in which murine bone marrow–derived MDSCs were cocultured with MC38 cells. Bottom left: Representative images of the migration of MDSCs toward YAP1-regulated MC38 cells. Right: Chart of the number of MDSCs that crossed the membrane in each group. ( N ) Left: Representative liver images of CRLM model mice in the four groups shown in the figure. Right: Chart of tumor numbers in the four groups. Scale bars, 100 μm (10X) and 20 μm (20X).

Article Snippet: The primary antibodies used for IHC, IF, and WB analyses were as follows: YAP1 (14074, CST, USA), YAP1 S127 (13008, CST), CXCL5 (DF9919, Affinity, USA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (2118, CST), LATS1 (3477, CST), p-LATS1 (9157, CST), lamin B1 (12987-1-AP, Proteintech, USA), connective tissue growth factor (CTGF; ab227180, Abcam, UK), Cystein Rich 61 (Cyr61; ab228592, Abcam), GLS (12855-1-AP, Proteintech), GOT1 (34423, CST), GLUL (80636, CST), PSAT1 (ab154055, Abcam), SLC1A5 (ab237704, Abcam), SLC7A5 (32683, CST), and SLC38A1 (28632-1-AP, Proteintech).

Techniques: RNA Sequencing, Immunohistochemistry, Over Expression, Knockdown, Transwell Migration Assay, Derivative Assay, Migration, Membrane

Journal: Science Advances

Article Title: Liver regeneration–associated hepatocellular YAP1 activation prevents colorectal cancer liver metastasis through glutamine competition

doi: 10.1126/sciadv.adw6926

Figure Lengend Snippet: ( A ) Gln levels in the tumors of CRLM mice (MC38/CT26 generated) in sham and 70% PHx groups. ( B ) WB analysis of YAP1 and YAP1 S127 levels in MC38 cells with different Gln levels. ( C ) qRT-PCR analysis of Ccn1 and Ccn2 levels in MC38 cells with different Gln levels. ( D ) WB analysis of YAP1, YAP1 S127 , and (P-)LATS1 levels in MC38 cells treated with different CB-839 concentrations. ( E ) WB analysis of YAP1 and YAP1 S127 levels in MC38 cells upon Gln deprivation and/or CB-839 treatment. ( F ) WB analysis of YAP1 and YAP1 S127 levels in the cytosol and nucleus of MC38 cells upon Gln deprivation and/or CB-839 treatment. ( G ) Cytometry analysis of relative ROS levels in MC38 cells treated with or without CB-839. MFI, mean fluorescence intensity. ( H ) WB analysis of YAP1, YAP1 S127 , and (P-)LATS1 levels in MC38 cells treated with CB-839 and/or subjected to Gln deprivation. ( I ) WB analysis of YAP1 and YAP1 S127 levels in MC38 cells treated with H 2 O 2 . ( J ) GSH levels in MC38 cells treated with or without BSO. ( K ) WB analysis of YAP1, YAP1 S127 , and (P-)LATS1 levels in MC38 cells treated with different BSO concentrations. ( L ) NADPH levels in MC38 cells treated with/without apocynin. ( M ) Cytometry analysis of relative ROS levels in MC38 cells treated with/without apocynin. ( N ) WB analysis of YAP1 and YAP1 S127 levels in MC38 cells treated with apocynin. ( O ) WB analysis of YAP1, YAP1S127, and CXCL5 levels in MC38 cells under the treatment of Gln deprivation or GLS inhibition with or without NAC treatment. ( P ) Representative images and chart of the number of tumors in the CRLM mice in the vehicle and CB-839 groups. ( Q ) WB analysis of YAP1 and YAP1 S127 levels in the tumors of the CRLM mice in each group.

Article Snippet: The primary antibodies used for IHC, IF, and WB analyses were as follows: YAP1 (14074, CST, USA), YAP1 S127 (13008, CST), CXCL5 (DF9919, Affinity, USA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (2118, CST), LATS1 (3477, CST), p-LATS1 (9157, CST), lamin B1 (12987-1-AP, Proteintech, USA), connective tissue growth factor (CTGF; ab227180, Abcam, UK), Cystein Rich 61 (Cyr61; ab228592, Abcam), GLS (12855-1-AP, Proteintech), GOT1 (34423, CST), GLUL (80636, CST), PSAT1 (ab154055, Abcam), SLC1A5 (ab237704, Abcam), SLC7A5 (32683, CST), and SLC38A1 (28632-1-AP, Proteintech).

Techniques: Generated, Quantitative RT-PCR, Cytometry, Fluorescence, Inhibition

Journal: Science Advances

Article Title: Liver regeneration–associated hepatocellular YAP1 activation prevents colorectal cancer liver metastasis through glutamine competition

doi: 10.1126/sciadv.adw6926

Figure Lengend Snippet: ( A ) Volcano plot of untargeted metabolomics analysis of metabolites in the tumor-adjacent livers of CRLM model mice in the sham and 70% PHx groups. ( B ) Relative levels of Gln in the tumor-adjacent livers of the CRLM model mice in sham, 30% PHx, and 70% PHx groups. ( C ) Levels of Gln and WB analysis of YAP1 S127 and nuclear YAP1 in the tumor-adjacent livers of the CRLM model mice in sham, 70% PHx, and 70% PHx with verteporfin groups. ( D ) qRT-PCR analysis of YAP1 downstream genes and glutamine metabolism–related genes levels in the tumor-adjacent livers of the CRLM model mice in sham, 30% PHx, and 70% PHx groups. ( E ) WB analysis of YAP1, YAP1 S127 , YAP1 downstream proteins, and glutamine metabolism–related proteins in the whole-cell lysates and nuclear fractions of the tumor-adjacent livers of the CRLM model mice in each group. ( F and G ) WB and qPCR analysis of YAP1, YAP1 downstream proteins, and glutamine metabolism–related proteins in the whole-cell lysates and nuclear fractions of AML12 cells with/without YAP1 overexpression. ( H ) Levels of Gln and glucose in AML12 cells with/without YAP1 overexpression. ( I and J ) WB and qPCR analysis of YAP1, YAP1 downstream proteins, and glutamine metabolism–related proteins in the whole-cell lysates and nuclei of AML12 cells with/without YAP1 knockdown. ( K ) Levels of Gln and glucose in AML12 cells with/without YAP1 knockdown.

Article Snippet: The primary antibodies used for IHC, IF, and WB analyses were as follows: YAP1 (14074, CST, USA), YAP1 S127 (13008, CST), CXCL5 (DF9919, Affinity, USA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (2118, CST), LATS1 (3477, CST), p-LATS1 (9157, CST), lamin B1 (12987-1-AP, Proteintech, USA), connective tissue growth factor (CTGF; ab227180, Abcam, UK), Cystein Rich 61 (Cyr61; ab228592, Abcam), GLS (12855-1-AP, Proteintech), GOT1 (34423, CST), GLUL (80636, CST), PSAT1 (ab154055, Abcam), SLC1A5 (ab237704, Abcam), SLC7A5 (32683, CST), and SLC38A1 (28632-1-AP, Proteintech).

Techniques: Quantitative RT-PCR, Over Expression, Knockdown

Journal: Science Advances

Article Title: Liver regeneration–associated hepatocellular YAP1 activation prevents colorectal cancer liver metastasis through glutamine competition

doi: 10.1126/sciadv.adw6926

Figure Lengend Snippet: ( A ) Representative IHC images of YAP1 expression patterns and scores indicating YAP1 levels in human CRLM-adjacent livers. ( B and C ) Survival and recurrence curves divided by liver YAP1 expression. ( D and E ) Chart showing the relationships between liver YAP1 expression and the number of tumors (D) and the largest tumor diameter (E). ( F ) Representative IHC images of YAP1 expression patterns and scores indicating YAP1 levels in the tumors of human patients with CRLM. ( G and H ) Survival curves of patients with CRLM divided by tumoral YAP1 expression. ( I and J ) Chart showing the correlation between tumor YAP1 expression and the number of tumors (I) and the largest tumor diameter (J). Statistically significant differences were identified via unpaired two-tailed Student’s t tests. ( K to N ) Representative IHC images of liver YAP1, tumor YAP1, and tumor CXCL5 in two standard patients (K) and the correlations between these markers (L to N). Scale bars, 200 μm (4X) and 20 μm (20X).

Article Snippet: The primary antibodies used for IHC, IF, and WB analyses were as follows: YAP1 (14074, CST, USA), YAP1 S127 (13008, CST), CXCL5 (DF9919, Affinity, USA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (2118, CST), LATS1 (3477, CST), p-LATS1 (9157, CST), lamin B1 (12987-1-AP, Proteintech, USA), connective tissue growth factor (CTGF; ab227180, Abcam, UK), Cystein Rich 61 (Cyr61; ab228592, Abcam), GLS (12855-1-AP, Proteintech), GOT1 (34423, CST), GLUL (80636, CST), PSAT1 (ab154055, Abcam), SLC1A5 (ab237704, Abcam), SLC7A5 (32683, CST), and SLC38A1 (28632-1-AP, Proteintech).

Techniques: Expressing, Two Tailed Test