rabbit anti p yap1  (Cell Signaling Technology Inc)

Journal: Oncology Reports

Article Title: Epigenetic silencing of JAM3 promotes laryngeal squamous cell carcinoma development by inhibiting the Hippo pathway

doi: 10.3892/or.2024.8861

Figure Lengend Snippet: JAM3 mediates laryngeal squamous cell carcinoma tumorigenesis through the Hippo pathway. (A) Protein levels of Flag, JAM3, p-LATS1 (Thr1079), LATS1, p-YAP1 (Ser127), YAP1 and β-actin in AMC-HN-8 and FD-LSC-1 cells with overexpression or knockdown of JAM3 were detected by western blotting. (B) Expression of YAP1 in AMC-HN-8 and FD-LSC-1 cells after transfection with the JAM3 overexpression plasmid or si-JAM3 were detected by confocal microscopy. Scale bar, 50 µm. Fluorescence ratio of YAP1 in the cytoplasm and nucleus of (C) AMC-HN-8 and (D) FD-LSC-1 cells was calculated by ImageJ and analyzed by two-tailed Student's t-test or one-way ANOVA. Data are presented as the mean ± SD of three independent experiments. *P<0.05 vs. Vec; # P<0.05, ## P<0.01, ### P<0.001, #### P<0.0001 vs. si-NC group. JAM3, junctional adhesion molecule 3; LATS1, large tumor suppressor kinase 1; NC, negative control; p-, phosphorylated; si, small interfering; Vec, p3×Flag-CMV-10 empty vector; YAP1, yes-associated protein 1.

Article Snippet: The membranes were then blocked in 10% non-fat milk (BD Biosciences) for 1.5 h at room temperature to prevent non-specific binding, and were incubated with primary antibodies targeting Flag (cat. no. F1804; 1:1,000; mouse; MilliporeSigma), JAM3 (cat. no. bs-11086R; 1:1,000; rabbit; BIOSS), large tumor suppressor kinase 1 (LATS1; cat. no. 17049-1-AP; 1:1,000; rabbit; Proteintech Group, Inc.), phosphorylated (p)-LATS1 (Thr1079) (cat. no. 28998-1-AP; 1:5,000; rabbit; Proteintech Group, Inc.), yes-associated protein 1 (YAP1; cat. no. 13584-1-AP; 1:5,000; rabbit; Proteintech Group, Inc.), p-YAP1 (Ser127) (cat. no. 13008S; 1:1,000; rabbit; Cell Signaling Technology, Inc.) and β-actin (cat. no. HC201-02; 1:2,000; mouse; TransGen Biotech Co., Ltd.) overnight at 4°C.

Techniques: Over Expression, Knockdown, Western Blot, Expressing, Transfection, Plasmid Preparation, Confocal Microscopy, Fluorescence, Two Tailed Test, Negative Control

Journal: Oncology Reports

Article Title: Epigenetic silencing of JAM3 promotes laryngeal squamous cell carcinoma development by inhibiting the Hippo pathway

doi: 10.3892/or.2024.8861

Figure Lengend Snippet: Knockdown of JAM3 promotes tumorigenicity of laryngeal squamous cell carcinoma cells in vivo . (A) Representative images of tumors in nude mice after subcutaneous injection of AMC-HN-8 cells transfected with si-NC and si-JAM3. (B) Tumor growth curve was plotted using xenograft tumor volume data. ***P<0.001 vs. si-NC. (C) Tumor weight was measured after tumor excision. *P<0.05 vs. si-NC group. (D) Relative expression levels of JAM3 in xenograft tumors as determined by reverse transcription-quantitative PCR analysis. Data are presented as the mean ± SD of three independent experiments. ****P<0.0001 vs. si-NC. (E) Hematoxylin and eosin staining of xenograft tumors. Immunohistochemistry staining of (F) JAM3, Ki-67 and YAP1, and (G) E-cadherin, N-cadherin and Vimentin in xenograft tumors. Scale bar, 20 µm. JAM3, junctional adhesion molecule 3; NC, negative control; si, small interfering; YAP1, yes-associated protein 1.

Article Snippet: The membranes were then blocked in 10% non-fat milk (BD Biosciences) for 1.5 h at room temperature to prevent non-specific binding, and were incubated with primary antibodies targeting Flag (cat. no. F1804; 1:1,000; mouse; MilliporeSigma), JAM3 (cat. no. bs-11086R; 1:1,000; rabbit; BIOSS), large tumor suppressor kinase 1 (LATS1; cat. no. 17049-1-AP; 1:1,000; rabbit; Proteintech Group, Inc.), phosphorylated (p)-LATS1 (Thr1079) (cat. no. 28998-1-AP; 1:5,000; rabbit; Proteintech Group, Inc.), yes-associated protein 1 (YAP1; cat. no. 13584-1-AP; 1:5,000; rabbit; Proteintech Group, Inc.), p-YAP1 (Ser127) (cat. no. 13008S; 1:1,000; rabbit; Cell Signaling Technology, Inc.) and β-actin (cat. no. HC201-02; 1:2,000; mouse; TransGen Biotech Co., Ltd.) overnight at 4°C.

Techniques: Knockdown, In Vivo, Injection, Transfection, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Staining, Immunohistochemistry, Negative Control

Journal: Oncology Reports

Article Title: Epigenetic silencing of JAM3 promotes laryngeal squamous cell carcinoma development by inhibiting the Hippo pathway

doi: 10.3892/or.2024.8861

Figure Lengend Snippet: Mechanism by which epigenetic silencing of JAM3 promotes LSCC development by inhibiting the Hippo pathway. JAM3, junctional adhesion molecule 3; LATS1, large tumor suppressor kinase 1; YAP1, yes-associated protein 1; p, phosphorylated; EMT, epithelial-mesenchymal transition.

Article Snippet: The membranes were then blocked in 10% non-fat milk (BD Biosciences) for 1.5 h at room temperature to prevent non-specific binding, and were incubated with primary antibodies targeting Flag (cat. no. F1804; 1:1,000; mouse; MilliporeSigma), JAM3 (cat. no. bs-11086R; 1:1,000; rabbit; BIOSS), large tumor suppressor kinase 1 (LATS1; cat. no. 17049-1-AP; 1:1,000; rabbit; Proteintech Group, Inc.), phosphorylated (p)-LATS1 (Thr1079) (cat. no. 28998-1-AP; 1:5,000; rabbit; Proteintech Group, Inc.), yes-associated protein 1 (YAP1; cat. no. 13584-1-AP; 1:5,000; rabbit; Proteintech Group, Inc.), p-YAP1 (Ser127) (cat. no. 13008S; 1:1,000; rabbit; Cell Signaling Technology, Inc.) and β-actin (cat. no. HC201-02; 1:2,000; mouse; TransGen Biotech Co., Ltd.) overnight at 4°C.

Techniques:

Journal: Molecular Therapy Oncology

Article Title: The YAP1-MAML2 fusion drives tumorigenesis and sustains tumor growth

doi: 10.1016/j.omton.2024.200900

Figure Lengend Snippet: Identification of YM fusion breakpoint and detection of endogenous YM fusion transcript and protein in ES-2 ovarian cancer cell line (A) Amplification of YAP1-MAML2 (YM) fragments using primer sets spanning YAP1 exons 3–5 and MAML2 exon 2. RT-PCR was performed using RNA from ES-2 cells, and amplified products were visualized by agarose gel electrophoresis followed by ethidium bromide staining. (B) Sequence of the fusion junction is shown. (C) Schematic representation of the fusion event, which results in the creation of a novel YM chimeric gene and a fusion protein comprising 328 aa from exons 1–5 of YAP1 (1–328 aa of YAP1) and 982 aa from exons 2–5 of MAML2 (172–1153 aa of MAML2). (D) Detection of endogenous YM protein in ES-2 cells using an anti-MAML2 C-terminal TAD antibody. H292 cells, which express a different fusion protein, CRTC1-MAML2, were used as a control. Note that cells also express native MAML2 proteins.

Article Snippet: The following antibodies were used: anti-GFP rabbit antibody (sc-8334) from Santa Cruz; anti-FLAG (PA1-984B) from Invitrogen; anti-β-Actin (A5316) from Sigma; and anti-MAML2 (4618), anti-YAP1 rabbit mAb (14074), anti-p-YAP1 (Ser397) rabbit mAb (13619), anti-pan-TEAD rabbit mAb (13295) and rabbit (DA1E) mAb IgG XP Isotype Control (3900), and anti-GAPDH (D16H11) XP rabbit mAb (5174) from Cell Signaling Technologies.

Techniques: Amplification, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Sequencing, Control

Journal: Molecular Therapy Oncology

Article Title: The YAP1-MAML2 fusion drives tumorigenesis and sustains tumor growth

doi: 10.1016/j.omton.2024.200900

Figure Lengend Snippet: The YM fusion exhibits nuclear speckles, interacts with the TEAD transcription factors, and activates YAP1/TEAD-responsive reporter (A) Expression of the GFP-YM construct was confirmed by transfection of COS7 cells with an empty vector or a vector expressing GFP-YM followed by western blotting using anti-MAML2 TAD and anti-GFP antibodies. (B) GFP-tagged YM exhibited a nuclear speckle pattern (top). The same cells were stained with DAPI to label the nuclei (bottom). Scale bar: 10 μm. (C) Immunofluorescence analysis of ES-2 MAML2 knockout (KO) cells, using anti-MAML2 TAD antibodies, followed by DAPI staining revealed that the endogenous YM fusion protein displays a pattern of nuclear dots (top). The specific MAML2 KO was confirmed via western blot (WB) analysis (bottom). (D) YM-TEAD protein interaction in ES-2 cells was revealed via coIP/WB analysis. Whole-cell protein lysates (750 μg) from parental ES-2 cells were subjected to immunoprecipitation (IP) with anti-TEAD or rabbit IgG (negative control) using Protein A/G beads. IP products were analyzed for TEAD and YM by western blotting. A total of 75 μg whole-cell protein lysate served as an input. (E) The YM fusion was able to activate a YAP-responsive promoter. 293FT cells were plated at 1 × 10 5 cells/well in 24-well plates overnight and transfected with 10 ng Renilla luciferase control vector, 200 ng YAP/TEAD-responsive firefly reporter vector (8xGTIIC-Luc), and 200 ng of pCMV2 empty vector, pCMV2-MAML2, pCMV2-YAP2, or pCMV2-YM fusion. After 48 h, luciferase assays were performed, and YAP1 promoter firefly luciferase activity was shown as fold activation relative to the basal level with an empty pCMV2 vector (n = 2, mean ± SD).

Article Snippet: The following antibodies were used: anti-GFP rabbit antibody (sc-8334) from Santa Cruz; anti-FLAG (PA1-984B) from Invitrogen; anti-β-Actin (A5316) from Sigma; and anti-MAML2 (4618), anti-YAP1 rabbit mAb (14074), anti-p-YAP1 (Ser397) rabbit mAb (13619), anti-pan-TEAD rabbit mAb (13295) and rabbit (DA1E) mAb IgG XP Isotype Control (3900), and anti-GAPDH (D16H11) XP rabbit mAb (5174) from Cell Signaling Technologies.

Techniques: Expressing, Construct, Transfection, Plasmid Preparation, Western Blot, Staining, Immunofluorescence, Knock-Out, Immunoprecipitation, Negative Control, Luciferase, Control, Activity Assay, Activation Assay

Journal: Molecular Therapy Oncology

Article Title: The YAP1-MAML2 fusion drives tumorigenesis and sustains tumor growth

doi: 10.1016/j.omton.2024.200900

Figure Lengend Snippet: The YM fusion has transforming activity (A) HEK293T cells were transfected with the expression constructs (pCMV2 vector, pCMV2-MAML2, pCMV2-YAP1, and pCMV2-YM), and protein expression was assessed by western blotting using the specified antibodies. (B) RK3E cells were transfected as described above and subsequently evaluated for colony formation at day 15 post-transfection using crystal violet staining.

Article Snippet: The following antibodies were used: anti-GFP rabbit antibody (sc-8334) from Santa Cruz; anti-FLAG (PA1-984B) from Invitrogen; anti-β-Actin (A5316) from Sigma; and anti-MAML2 (4618), anti-YAP1 rabbit mAb (14074), anti-p-YAP1 (Ser397) rabbit mAb (13619), anti-pan-TEAD rabbit mAb (13295) and rabbit (DA1E) mAb IgG XP Isotype Control (3900), and anti-GAPDH (D16H11) XP rabbit mAb (5174) from Cell Signaling Technologies.

Techniques: Activity Assay, Transfection, Expressing, Construct, Plasmid Preparation, Western Blot, Staining

Journal: Molecular Therapy Oncology

Article Title: The YAP1-MAML2 fusion drives tumorigenesis and sustains tumor growth

doi: 10.1016/j.omton.2024.200900

Figure Lengend Snippet: RNA-seq analysis of YM target genes and pathways (A) Differentially expressed genes in ES-2 cells after the depletion of YM/MAML2 are shown in Volcano plot. A few representative YAP1 target genes were indicated. (B and C) The real-time qPCR assays of YM-regulated genes identified from RNA-seq study were conducted using YM/MAML2 knockdown cells (shM2-1 and shM2-3) and the MAML2-KO and their respective control cells (ES-2 shCtl and ES-2 sgCtl) (n = 3, mean ± SD). (D) IPA analysis identified several transcription regulators of the transcriptomic response to YM/MAML2 knockdown. The changes in the activation or inhibition status of transcription regulators were plotted based on their Z scores. A positive Z score indicates activation, while a negative Z score denotes inhibition. (E) GSEA showed that the YAP conserved signature, MYC target, and E2F target gene sets were negatively enriched, whereas the inflammatory response genes were positively enriched in fusion-depleted cells compared to controls.

Article Snippet: The following antibodies were used: anti-GFP rabbit antibody (sc-8334) from Santa Cruz; anti-FLAG (PA1-984B) from Invitrogen; anti-β-Actin (A5316) from Sigma; and anti-MAML2 (4618), anti-YAP1 rabbit mAb (14074), anti-p-YAP1 (Ser397) rabbit mAb (13619), anti-pan-TEAD rabbit mAb (13295) and rabbit (DA1E) mAb IgG XP Isotype Control (3900), and anti-GAPDH (D16H11) XP rabbit mAb (5174) from Cell Signaling Technologies.

Techniques: RNA Sequencing, Knockdown, Control, Activation Assay, Inhibition

Journal: Molecular Therapy Oncology

Article Title: The YAP1-MAML2 fusion drives tumorigenesis and sustains tumor growth

doi: 10.1016/j.omton.2024.200900

Figure Lengend Snippet: The YM-positive ES-2 cells were sensitive to YAP1 inhibitors (A and B) Cells were cultured in 24-well plates (4 × 10 4 cells per well for ES-2 and 7.5 × 10 4 cells per well for Heya-8 and H3118) overnight and then treated with YAP1 inhibitors (CA3 or VPF) at the indicated concentrations for 48 h. Cells were stained using crystal violet (A), and viable cell numbers were determined by Trypan blue assay (B) ( n = 2, mean ± SD). (C) ES-2 cells were seeded at 500 cells per well in 12-well plates and treated with CA3 or VPF at the indicated concentrations ( n = 3, mean ± SD). After 14 days of culture, colonies were fixed and stained with crystal violet. The colony areas were quantified using ImageJ and presented as a percentage of the colony area relative to the corresponding DMSO-treated control groups. ∗<0.05, ∗∗<0.005, ∗∗∗,0.0005.

Article Snippet: The following antibodies were used: anti-GFP rabbit antibody (sc-8334) from Santa Cruz; anti-FLAG (PA1-984B) from Invitrogen; anti-β-Actin (A5316) from Sigma; and anti-MAML2 (4618), anti-YAP1 rabbit mAb (14074), anti-p-YAP1 (Ser397) rabbit mAb (13619), anti-pan-TEAD rabbit mAb (13295) and rabbit (DA1E) mAb IgG XP Isotype Control (3900), and anti-GAPDH (D16H11) XP rabbit mAb (5174) from Cell Signaling Technologies.

Techniques: Cell Culture, Staining, Control