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: British journal of cancer

Article Title: Recurrent somatic mutations of FAT family cadherins induce an aggressive phenotype and poor prognosis in anaplastic large cell lymphoma.

doi: 10.1038/s41416-024-02881-7

Figure Lengend Snippet: Fig. 3 Loss of FAT4 activates YAP1 and β-catenin signaling. a Phosphorylation of relevant signaling targets in shFAT4 vs shNT cells, determined by phospho-protein array. b Western blot analysis shows simultaneous de-phosphorylation of β-catenin (p-Ser33/37/Thr41) and YAP1 (p-Ser127), and increased STAT3 expression, in shFAT4 cells. c Confocal microscopy showing increased β-catenin and YAP1 nuclear localization in shFAT4 cells (green: β-catenin and YAP1; blue: Hoechst). d Quantification of the nuclear fraction from the data obtained by confocal microscopy (n = 25). e TOP-Flash luciferase assay. f Expression of the indicated transcripts by qPCR. g Sensitivity of shNT and shFAT4 cells to PKF115-584, a blocker of β-catenin/Tcf4 interaction. h Effects of crizotinib/PKF115-584 combinations on cells viability. i Heatmap of Bliss synergy scores obtained by crizotinib combinations with β-catenin or YAP1 inhibitors, in shFAT4 cells. *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: Primary antibodies used in this study were as follows: β-catenin (BDbiosciences, cat# 610154; dilution (dil) 1:1000), phospho-β-catenin (Ser33/Ser37/Thr41; Cell Signaling Technology [CST], cat# 9561; dil 1:1000), YAP1 (Santa Cruz Biotechnology, cat# sc-376830; dil 1:500), phospho-YAP1 (Ser127; CST, cat# 4911; dil 1:1000), STAT3 (CST, clone D3Z2G, cat# 12640; dil 1:1000), actin (Sigma, cat# A2066; dil 1:2000), GAPDH (AbCam, cat# ab9485; dil 1:500), FLAG-tag (Sigma, clone M2, cat# F3165; dil 1:1000), HA-tag (CST, clone C29F4, cat# 3724; dil 1:1000), PRMT5 (CST, clone D5P2T, cat# 79998; dil 1:1000), DBC1 (CST, cat# 5857; dil 1:1000), FAT4 (Novus biological; cat# NBPI-78381; dil 1:150; used for immunohistochemistry following the protocol recommended by the manufacturer).

Techniques: Phospho-proteomics, Protein Array, Western Blot, De-Phosphorylation Assay, Expressing, Confocal Microscopy, Luciferase

Journal: British journal of cancer

Article Title: Recurrent somatic mutations of FAT family cadherins induce an aggressive phenotype and poor prognosis in anaplastic large cell lymphoma.

doi: 10.1038/s41416-024-02881-7

Figure Lengend Snippet: Fig. 6 Schematic overview of the findings. NPM::ALK fusion kinase activates β-catenin signaling and induces cytoskeleton remodeling. Loss of FAT4-mediated inhibition activates YAP1, which potentiates both F-actin and β-catenin effects. YAP1 is itself further activated by β-catenin and cell motility, in a positive loop that leads to enhanced cell growth and migration. Created with BioRender.

Article Snippet: Primary antibodies used in this study were as follows: β-catenin (BDbiosciences, cat# 610154; dilution (dil) 1:1000), phospho-β-catenin (Ser33/Ser37/Thr41; Cell Signaling Technology [CST], cat# 9561; dil 1:1000), YAP1 (Santa Cruz Biotechnology, cat# sc-376830; dil 1:500), phospho-YAP1 (Ser127; CST, cat# 4911; dil 1:1000), STAT3 (CST, clone D3Z2G, cat# 12640; dil 1:1000), actin (Sigma, cat# A2066; dil 1:2000), GAPDH (AbCam, cat# ab9485; dil 1:500), FLAG-tag (Sigma, clone M2, cat# F3165; dil 1:1000), HA-tag (CST, clone C29F4, cat# 3724; dil 1:1000), PRMT5 (CST, clone D5P2T, cat# 79998; dil 1:1000), DBC1 (CST, cat# 5857; dil 1:1000), FAT4 (Novus biological; cat# NBPI-78381; dil 1:150; used for immunohistochemistry following the protocol recommended by the manufacturer).

Techniques: Inhibition, Migration

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: CircGPRC5A enhances colorectal cancer progress by stabilizing PPP1CA and inducing YAP dephosphorylation

doi: 10.1186/s13046-023-02915-7

Figure Lengend Snippet: CircGPRC5A can cause dephosphorylation of YAP-ser127 and YAP-ser109 by stabilizing the PPP1CA protein. A Western blotting was used to examine PPP1CA protein after silencing circGPRC5A. B Phosphoproteomics revealed 4,170 phosphopeptides, 4,793 phosphorylation sites, and 2,390 phosphoproteins. C The pie chart illustrates the types of phosphorylated amino acids from quantitative phosphoproteomics analysis. D Upregulated and downregulated protein phosphorylation levels from quantitative phosphoproteomics analysis. E Bubble chart depicting the deltaP_value and phosphorylation site number. F , G Western blotting was used to examine YAP phosphorylation after silencing and overexpressing PPP1CA or circGPRC5A. H – I The effects of YAP dephosphorylation via PPP1CA on circGPRC5A-induced changes were detected by western blotting

Article Snippet: The primary antibodies used were PPP1CA (sc-7482, Santa Cruz Biotechnology), Ub (#3936S CST), UBA1 (ab180125, Abcam), FBXO2 (ab133717, Abcam), YAP-Ser127 (AP0489, ABclonal), YAP-Ser109 (YP1553, Immunomy), YAP-Ser61(#75784S, CST), YAP (A21216, ABclonal), Pan Phospho-Serine/Threonine (T91067S, Abmart), and GAPDH (10,494–1-AP, Proteintech).

Techniques: De-Phosphorylation Assay, Western Blot, Phospho-proteomics

Journal: Oncogenesis

Article Title: PKC-independent PI3K signalling diminishes PKC inhibitor sensitivity in uveal melanoma

doi: 10.1038/s41389-024-00511-8

Figure Lengend Snippet: A Accumulation of MAPK, PI3K/AKT, YAP and PKC signalling effectors including total and phosphorylated DUSP6, MARCKS, ERK, YAP, S6 and AKT 24 h after treatment with vehicle control (−) or 10 nM Trametinib (+). Western blot analyses were performed using three independent biological replicates ( n = 3). kD kilodalton. REVERT total protein loading stain is shown in Supplementary Fig. . B Expression levels of pS6 and DUSP6 (normalised to total S6 or REVERT staining, respectively) in vehicle control or 10 nM Trametinib-treated GNAQ/GNA11 -mutant (solid dot) or wild-type (crossed dot) UM cell lines. Data derived from three independent biological experiments ( n = 3), and P values were calculated using paired t tests. ns not significant. C Percentage of cells undergoing S-phase inhibition (dotted line set at 50% S-phase inhibition) and change in % sub-G1 (dotted line set at 30% sub-G1) in GNAQ/GNA11 -mutant (solid circle) or wild-type (crossed circle) UM cell lines treated with 5 µM IDE196 (data derived from ), 10 nM Trametinib or 2 µM BEZ235. Data derived from three independent biological experiments ( n = 3).

Article Snippet: Membranes were incubated at 4 °C overnight in primary antibodies diluted in Intercept Blocking Buffer (TBS) (Li-Cor, Lincoln, NE, USA) or Odyssey Blocking Buffer (Li-Cor) with Tween 20 (0.05%), as follows: total MARCKS (1:1000, 2C2, WH0004082M6, Sigma-Aldrich), phosphorylated MARCKS (pMARCKS; Ser 152/156 , 1:1000, 2741 S, Cell Signaling Technology, Danvers, MA, USA), DUSP6 (1:250 or 1:1000, EPR129Y, ab76310, Abcam, Cambridge, UK), total AKT (1:1000, 40D4, 2920S, Cell Signaling Technology), phosphorylated AKT (pAKT; Ser 473 , 1:100, D9E, 4060S, Cell Signaling Technology), pAKT (Ser 473 , 1:500, 736E11, 3787, Cell Signaling Technology), total ribosomal S6 (1:500, 54D2, 2317 S, Cell Signaling Technology), phosphorylated ribosomal S6 (pS6; Ser 235/236 , 1:1000, 2F9, 4856S, Cell Signaling Technology), total YAP (1:500, 1A12, 12395S, Cell Signaling Technology), phosphorylated YAP (pYAP; Ser 127 , 1:2000, 4911, Cell Signaling Technology), total ERK (1:2 000, 137F5, 4695S, Cell Signaling Technology), phosphorylated ERK (pERK; Tyr 204 , 1:250, E-4, SC-7383, Santa Cruz, Dallas, TX, USA), and MEK1/2 (1:500, L38C12, 4694S, Cell Signaling Technology).

Techniques: Control, Western Blot, Staining, Expressing, Mutagenesis, Derivative Assay, Inhibition

Journal: Oncogenesis

Article Title: PKC-independent PI3K signalling diminishes PKC inhibitor sensitivity in uveal melanoma

doi: 10.1038/s41389-024-00511-8

Figure Lengend Snippet: A Fold change in DUSP6 and pS6 expression (normalised log 2 protein expression in drug-treated – normalised log 2 protein expression in control-treated cells) in GNAQ/GNA11 -mutant (solid circle) and wild-type (crossed circle) UM cell lines treated with 5 µM IDE196, 10 nM Trametinib, or 2 µM BEZ235. Data compared using one-way ANOVA with the Geisser–Greenhouse correction and Tukey’s multiple comparison test, adjusted P values are shown. Data derived from three independent biological experiments ( n = 3, mean ± SD). B Accumulation of MAPK, PI3K, YAP and PKC signalling effectors, including total and phosphorylated DUSP6, MARCKS, ERK, YAP, S6 and AKT 24 h after treatment with vehicle control (−) or 2 µM BEZ235 (+). Western blot analyses were performed using three independent biological replicates ( n = 3). kD kilodalton. REVERT total protein loading stain is shown in Supplementary Fig. .

Article Snippet: Membranes were incubated at 4 °C overnight in primary antibodies diluted in Intercept Blocking Buffer (TBS) (Li-Cor, Lincoln, NE, USA) or Odyssey Blocking Buffer (Li-Cor) with Tween 20 (0.05%), as follows: total MARCKS (1:1000, 2C2, WH0004082M6, Sigma-Aldrich), phosphorylated MARCKS (pMARCKS; Ser 152/156 , 1:1000, 2741 S, Cell Signaling Technology, Danvers, MA, USA), DUSP6 (1:250 or 1:1000, EPR129Y, ab76310, Abcam, Cambridge, UK), total AKT (1:1000, 40D4, 2920S, Cell Signaling Technology), phosphorylated AKT (pAKT; Ser 473 , 1:100, D9E, 4060S, Cell Signaling Technology), pAKT (Ser 473 , 1:500, 736E11, 3787, Cell Signaling Technology), total ribosomal S6 (1:500, 54D2, 2317 S, Cell Signaling Technology), phosphorylated ribosomal S6 (pS6; Ser 235/236 , 1:1000, 2F9, 4856S, Cell Signaling Technology), total YAP (1:500, 1A12, 12395S, Cell Signaling Technology), phosphorylated YAP (pYAP; Ser 127 , 1:2000, 4911, Cell Signaling Technology), total ERK (1:2 000, 137F5, 4695S, Cell Signaling Technology), phosphorylated ERK (pERK; Tyr 204 , 1:250, E-4, SC-7383, Santa Cruz, Dallas, TX, USA), and MEK1/2 (1:500, L38C12, 4694S, Cell Signaling Technology).

Techniques: Expressing, Control, Mutagenesis, Comparison, Derivative Assay, Western Blot, Staining

Journal: Oncogenesis

Article Title: PKC-independent PI3K signalling diminishes PKC inhibitor sensitivity in uveal melanoma

doi: 10.1038/s41389-024-00511-8

Figure Lengend Snippet: A Accumulation of MAPK, PI3K/AKT, YAP and PKC signalling effectors including total and phosphorylated DUSP6, MARCKS, ERK, YAP, S6 and AKT 24 h after treatment with vehicle control (−) or 10 nM Trametinib (+). Western blot analyses were performed using three independent biological replicates ( n = 3). kD kilodalton. REVERT total protein loading stain is shown in Supplementary Fig. . B Expression levels of pS6 and DUSP6 (normalised to total S6 or REVERT staining, respectively) in vehicle control or 10 nM Trametinib-treated GNAQ/GNA11 -mutant (solid dot) or wild-type (crossed dot) UM cell lines. Data derived from three independent biological experiments ( n = 3), and P values were calculated using paired t tests. ns not significant. C Percentage of cells undergoing S-phase inhibition (dotted line set at 50% S-phase inhibition) and change in % sub-G1 (dotted line set at 30% sub-G1) in GNAQ/GNA11 -mutant (solid circle) or wild-type (crossed circle) UM cell lines treated with 5 µM IDE196 (data derived from ), 10 nM Trametinib or 2 µM BEZ235. Data derived from three independent biological experiments ( n = 3).

Article Snippet: Membranes were incubated at 4 °C overnight in primary antibodies diluted in Intercept Blocking Buffer (TBS) (Li-Cor, Lincoln, NE, USA) or Odyssey Blocking Buffer (Li-Cor) with Tween 20 (0.05%), as follows: total MARCKS (1:1000, 2C2, WH0004082M6, Sigma-Aldrich), phosphorylated MARCKS (pMARCKS; Ser 152/156 , 1:1000, 2741 S, Cell Signaling Technology, Danvers, MA, USA), DUSP6 (1:250 or 1:1000, EPR129Y, ab76310, Abcam, Cambridge, UK), total AKT (1:1000, 40D4, 2920S, Cell Signaling Technology), phosphorylated AKT (pAKT; Ser 473 , 1:100, D9E, 4060S, Cell Signaling Technology), pAKT (Ser 473 , 1:500, 736E11, 3787, Cell Signaling Technology), total ribosomal S6 (1:500, 54D2, 2317 S, Cell Signaling Technology), phosphorylated ribosomal S6 (pS6; Ser 235/236 , 1:1000, 2F9, 4856S, Cell Signaling Technology), total YAP (1:500, 1A12, 12395S, Cell Signaling Technology), phosphorylated YAP (pYAP; Ser 127 , 1:2000, 4911, Cell Signaling Technology), total ERK (1:2 000, 137F5, 4695S, Cell Signaling Technology), phosphorylated ERK (pERK; Tyr 204 , 1:250, E-4, SC-7383, Santa Cruz, Dallas, TX, USA), and MEK1/2 (1:500, L38C12, 4694S, Cell Signaling Technology).

Techniques: Control, Western Blot, Staining, Expressing, Mutagenesis, Derivative Assay, Inhibition

Journal: Oncogenesis

Article Title: PKC-independent PI3K signalling diminishes PKC inhibitor sensitivity in uveal melanoma

doi: 10.1038/s41389-024-00511-8

Figure Lengend Snippet: A Fold change in DUSP6 and pS6 expression (normalised log 2 protein expression in drug-treated – normalised log 2 protein expression in control-treated cells) in GNAQ/GNA11 -mutant (solid circle) and wild-type (crossed circle) UM cell lines treated with 5 µM IDE196, 10 nM Trametinib, or 2 µM BEZ235. Data compared using one-way ANOVA with the Geisser–Greenhouse correction and Tukey’s multiple comparison test, adjusted P values are shown. Data derived from three independent biological experiments ( n = 3, mean ± SD). B Accumulation of MAPK, PI3K, YAP and PKC signalling effectors, including total and phosphorylated DUSP6, MARCKS, ERK, YAP, S6 and AKT 24 h after treatment with vehicle control (−) or 2 µM BEZ235 (+). Western blot analyses were performed using three independent biological replicates ( n = 3). kD kilodalton. REVERT total protein loading stain is shown in Supplementary Fig. .

Article Snippet: Membranes were incubated at 4 °C overnight in primary antibodies diluted in Intercept Blocking Buffer (TBS) (Li-Cor, Lincoln, NE, USA) or Odyssey Blocking Buffer (Li-Cor) with Tween 20 (0.05%), as follows: total MARCKS (1:1000, 2C2, WH0004082M6, Sigma-Aldrich), phosphorylated MARCKS (pMARCKS; Ser 152/156 , 1:1000, 2741 S, Cell Signaling Technology, Danvers, MA, USA), DUSP6 (1:250 or 1:1000, EPR129Y, ab76310, Abcam, Cambridge, UK), total AKT (1:1000, 40D4, 2920S, Cell Signaling Technology), phosphorylated AKT (pAKT; Ser 473 , 1:100, D9E, 4060S, Cell Signaling Technology), pAKT (Ser 473 , 1:500, 736E11, 3787, Cell Signaling Technology), total ribosomal S6 (1:500, 54D2, 2317 S, Cell Signaling Technology), phosphorylated ribosomal S6 (pS6; Ser 235/236 , 1:1000, 2F9, 4856S, Cell Signaling Technology), total YAP (1:500, 1A12, 12395S, Cell Signaling Technology), phosphorylated YAP (pYAP; Ser 127 , 1:2000, 4911, Cell Signaling Technology), total ERK (1:2 000, 137F5, 4695S, Cell Signaling Technology), phosphorylated ERK (pERK; Tyr 204 , 1:250, E-4, SC-7383, Santa Cruz, Dallas, TX, USA), and MEK1/2 (1:500, L38C12, 4694S, Cell Signaling Technology).

Techniques: Expressing, Control, Mutagenesis, Comparison, Derivative Assay, Western Blot, Staining