Journal: Developmental cell

Article Title: The Hippo pathway regulates Wnt/beta-catenin signaling.

doi: 10.1016/j.devcel.2010.03.007

Figure Lengend Snippet: Figure 1. TAZ is a Negative Regulator of the Wnt/b-Catenin Pathway (A) TAZ knockdown enhances Wnt3A-induced activity of the TOPflash reporter. TOPflash reporter activity was measured in HEK293T cells transfected with increasing amounts (25 nM or 50 nM) of pooled or single TAZ siRNA (siTAZ) or a scrambled control (siCTL), and then treated with control or Wnt3A-conditioned media. (B) siRNA-resistant TAZ rescues enhanced Wnt signaling resulting from TAZ knockdown. TOPflash and FOPflash reporter activity was measured in HEK293T cells transfected with siCTL or siTAZ expressing very low levels of siRNA-resistant mouse Taz and after treatment with control of Wnt3A-conditioned media. The relative levels of mouse and human TAZ were measured by quantitative RT-PCR in parallel samples. (C) TAZ knockdown induces Wnt3A-dependent target gene expression. The expression of NKD1, TNFRSF19, and AXIN2 was determined by qPCR from MDA- MB-231 cells transfected with siTAZ or siCTL and treated for 4 or 16 hr with control or Wnt3A-conditioned media as indicated. (D) TAZ knockdown stabilizes b-Catenin levels. MDA-MB-231 cells were transfected with siTAZ or siCTL and treated with control or Wnt3A-conditioned media for 4 hr, and total cell lysates were subsequently analyzed for b-Catenin levels by immunoblotting. (E) Knockdown of TAZ induces nuclear b-Catenin accumulation. MDA-MB-231 cells were transfected with siTAZ or siCTL and treated with control or Wnt3A- conditioned media for 2 hr. Cells were then analyzed for endogenous b-Catenin localization by immunofluorescent microscopy. The scale bar represents 10 mM. The percentage of cells displaying high nuclear/cytoplasmic variance of b-Catenin signal is shown. Data show that after Wnt3A treatment, siTAZ-treated cells displayed high nuclear/cytoplasmic variance (p < 0.0001).

Article Snippet: Immunoprecipitation (IP) and immunoblotting (IB) were carried out with the following antibodies: Flag (Sigma #F3165; 1 mg/IP, 1:2000 IB); T7 (Novagen #69522-3; 1 mg/IP, 1:10,000 IB); HA (Roche #11867431001; 1:1000 IB); TAZ (BD Biosciences #560235; 3 mg/IP, 1:1000 IB); TAZ (Novus #NB600-220; 1:1000 IB); CK13 (BD Biosciences #610445; 1:2000 IB); b-Catenin (BD Biosci- Deve ences #610153; 1:1000 IB); Actin (Sigma # A2066; 1:2000 IB); DVL2 (Cell Signaling Technology #3224; 1:1000 IB); LATS1 (Bethyl #A300-478A; 1:1000 IB); MST1 (BD biosciences #77120; 1:1000 IB); MST2 (Novus #NB10079800; 1:1000 IB).

Techniques: Knockdown, Activity Assay, Transfection, Control, Expressing, Quantitative RT-PCR, Targeted Gene Expression, Western Blot, Microscopy

Journal: Developmental cell

Article Title: The Hippo pathway regulates Wnt/beta-catenin signaling.

doi: 10.1016/j.devcel.2010.03.007

Figure Lengend Snippet: Figure 3. TAZ Binds to Dishevelled (A) TAZ interacts with DVLs. HEK293T cells were transfected with HA-TAZ and Flag-DVL1, -2, or -3. After anti-Flag immunoprecipitation, the presence of TAZ was detected by anti-HA immunoblotting. (B) Interaction of endogenous TAZ and DVL2. Cell lysates from MDA-MB-231 cells were subjected to immunoprecipitation with anti-TAZ antibody or control IgG, and the presence of DVL2 was examined by anti-DVL2 immunoblotting. (C) DVL2 interacts with the WW domain and PDZ-binding motif of TAZ. HEK293T cells were cotransfected with the indicated Flag-TAZ WT or mutant constructs and DVL2 tagged with Firefly luciferase. Cell lysates were subjected to anti-Flag immunoprecipitation, and the presence of DVL2 was assessed by luciferase assay. (D) TAZ interacts with the PY motif and PDZ domain of DVL2. HEK293T cells were cotransfected with the indicated Flag-DVL2 WT or mutant constructs and HA-TAZ. Cell lysates were subjected to anti-Flag immunoprecipitation, and the presence of TAZ was assessed by anti-HA immunoblotting.

Article Snippet: Immunoprecipitation (IP) and immunoblotting (IB) were carried out with the following antibodies: Flag (Sigma #F3165; 1 mg/IP, 1:2000 IB); T7 (Novagen #69522-3; 1 mg/IP, 1:10,000 IB); HA (Roche #11867431001; 1:1000 IB); TAZ (BD Biosciences #560235; 3 mg/IP, 1:1000 IB); TAZ (Novus #NB600-220; 1:1000 IB); CK13 (BD Biosciences #610445; 1:2000 IB); b-Catenin (BD Biosci- Deve ences #610153; 1:1000 IB); Actin (Sigma # A2066; 1:2000 IB); DVL2 (Cell Signaling Technology #3224; 1:1000 IB); LATS1 (Bethyl #A300-478A; 1:1000 IB); MST1 (BD biosciences #77120; 1:1000 IB); MST2 (Novus #NB10079800; 1:1000 IB).

Techniques: Transfection, Immunoprecipitation, Western Blot, Control, Binding Assay, Mutagenesis, Construct, Luciferase

Journal: Developmental cell

Article Title: The Hippo pathway regulates Wnt/beta-catenin signaling.

doi: 10.1016/j.devcel.2010.03.007

Figure Lengend Snippet: Figure 4. TAZ Inhibits DVL2 Phosphorylation by CK1d/3 (A and B) TAZ knockdown induces CK1d/3-mediated DVL2 phosphorylation. (A) MDA-MB-231 cells were transfected with siCTL or siTAZ, treated with or without the CK1d/3 inhibitor IC261 for 1 hr, and then treated with control or Wnt3A-conditioned media for 4 hr. Cell lysates were then analyzed by immunoblotting with the indicated antibodies. (B) MDA-MB-231 cells were transfected with 50 nM siCTL or siTAZ, or 50 nM of total siRNA comprised of combinations of TAZ, CK1d, and CK13, as indicated. Cells were treated with control or Wnt3A-conditioned media for 4 hr, lysed, and analyzed by immunoblotting. (C) CK13 knockdown inhibits the enhanced TOPflash activity caused by TAZ knockdown. TOPflash activity was measured in HEK293T cells transfected with the indicated siRNAs, and then treated with control or Wnt3A-conditioned media. Data from a representative experiment are plotted as the mean of three replicates plus standard deviation. Knockdown was confirmed by immunoblotting. (D) TAZ overexpression inhibits the interaction of DVL2 with CK13. HEK293T cells were transfected with T7-DVL2, Flag-TAZ, and decreasing amounts of Flag- CK13. Interactions were detected by anti-T7 immunoprecipitation followed by anti-Flag immunoblotting. CK13 levels were quantitated with ImageQuant software (top panel). (E) A DVL2-binding mutant of TAZ does not inhibit the interaction of DVL2 with CK13. Cells were transfected with Firefly luciferase (Luc) or with CK13 tagged with Firefly luciferase (CK1-Luc) along with Flag-tagged WT or mutant (1–393, DWW) TAZ. The interaction between DVL2 and CK13 was detected by immunoprecip- itation of endogenous DVL2 followed by a luciferase assay. (F) WT TAZ, but not the DVL2-binding mutant nor the nuclear-localized TAZ-S89A, rescue the enhanced Wnt signaling resulting from TAZ knockdown. TOPflash reporter activity after treatment with control or Wnt3A-conditioned media was measured in HEK293T cells transfected with siCTL or siTAZ expressing low levels of siRNA-resistant mouse WT Taz, Taz (1-393, DWW), or Taz (S89A). Data from a representative experiment are plotted as the mean of three replicates plus stan- dard deviation.

Article Snippet: Immunoprecipitation (IP) and immunoblotting (IB) were carried out with the following antibodies: Flag (Sigma #F3165; 1 mg/IP, 1:2000 IB); T7 (Novagen #69522-3; 1 mg/IP, 1:10,000 IB); HA (Roche #11867431001; 1:1000 IB); TAZ (BD Biosciences #560235; 3 mg/IP, 1:1000 IB); TAZ (Novus #NB600-220; 1:1000 IB); CK13 (BD Biosciences #610445; 1:2000 IB); b-Catenin (BD Biosci- Deve ences #610153; 1:1000 IB); Actin (Sigma # A2066; 1:2000 IB); DVL2 (Cell Signaling Technology #3224; 1:1000 IB); LATS1 (Bethyl #A300-478A; 1:1000 IB); MST1 (BD biosciences #77120; 1:1000 IB); MST2 (Novus #NB10079800; 1:1000 IB).

Techniques: Phospho-proteomics, Knockdown, Transfection, Control, Western Blot, Activity Assay, Standard Deviation, Over Expression, Immunoprecipitation, Software, Binding Assay, Mutagenesis, Luciferase, Expressing

Journal: Developmental cell

Article Title: The Hippo pathway regulates Wnt/beta-catenin signaling.

doi: 10.1016/j.devcel.2010.03.007

Figure Lengend Snippet: Figure 5. The MST/LATS Pathway Inhibits Wnt/b-Catenin Signaling (A) Interaction of endogenous TAZ and LATS1. Cell lysates from MDA-MB-231 cells were subjected to immunoprecipitation with anti-TAZ antibody or control IgG, and the presence of LATS1 was detected by immunoblotting. (B) Loss of MST/LATS expression enhances Wnt3A-induced transcriptional responses. TOPflash reporter activity was measured in HEK293T cells transfected with siRNAs targeting LATS1, LATS2, MST1, or MST2, as indicated, and then incubated with control or Wnt3A-conditioned media. Data from a representative experiment are plotted as the mean of three replicates plus standard deviation. Immunoblots from total cell lysates were performed to confirm knockdown. Knockdown of LATS2 was confirmed by RT-PCR (data not shown). (C) MST/LATS knockdown enhances Wnt3A-dependent AXIN2 expression. The expression of AXIN2 was determined by qPCR from MDA-MB-231 cells trans- fected with siCTL, siLATS1, or siMST2, and then treated with control or Wnt3A-conditioned media for 4 hr. (D and E) (D) MST/LATS overexpression inhibits Wnt3A-induced TOPflash activity. HEK293T cells were cotransfected with the TOPflash reporter together with LATS1 and 0.05 mg or 0.1 mg MST2 or 0.1 mg MST2 (KR) as indicated, and then treated with control or Wnt3A conditioned media. (E) TAZ knockdown suppresses the inhibitory effect of MST/LATS overexpression on Wnt signaling. HEK293T cells were transfected with the TOPflash reporter, siCTL or siTAZ, and with LATS1 and MST2, and then treated with control or Wnt3A-conditioned media. (D and E) Data from representative experiments are plotted as the mean of three replicates plus standard deviation.

Article Snippet: Immunoprecipitation (IP) and immunoblotting (IB) were carried out with the following antibodies: Flag (Sigma #F3165; 1 mg/IP, 1:2000 IB); T7 (Novagen #69522-3; 1 mg/IP, 1:10,000 IB); HA (Roche #11867431001; 1:1000 IB); TAZ (BD Biosciences #560235; 3 mg/IP, 1:1000 IB); TAZ (Novus #NB600-220; 1:1000 IB); CK13 (BD Biosciences #610445; 1:2000 IB); b-Catenin (BD Biosci- Deve ences #610153; 1:1000 IB); Actin (Sigma # A2066; 1:2000 IB); DVL2 (Cell Signaling Technology #3224; 1:1000 IB); LATS1 (Bethyl #A300-478A; 1:1000 IB); MST1 (BD biosciences #77120; 1:1000 IB); MST2 (Novus #NB10079800; 1:1000 IB).

Techniques: Immunoprecipitation, Control, Western Blot, Expressing, Activity Assay, Transfection, Incubation, Standard Deviation, Knockdown, Reverse Transcription Polymerase Chain Reaction, Over Expression

Journal: Developmental cell

Article Title: The Hippo pathway regulates Wnt/beta-catenin signaling.

doi: 10.1016/j.devcel.2010.03.007

Figure Lengend Snippet: Figure 6. LATS1 Activity Promotes a Cytoplasmic Role for TAZ in the Inhibition of Wnt/b-Catenin Signaling (A) Abrogation of LATS1 expression induces TAZ nuclear accumulation and stimulates nuclear b-Catenin localization in response to Wnt3A. MDA-MB-231 cells were transfected with siCTL or siLATS1, and then treated with control or Wnt3A-conditioned media for 2 hr. Cells were then analyzed for endogenous TAZ and b-Catenin localization by immunofluorescent microscopy. The cell nuclei were visualized by DAPI staining. The scale bar represents 10 mM. The percentage of cells displaying high nuclear/cytoplasmic variance of b-Catenin signal is shown. Data show that after Wnt3A treatment, siLATS1-treated cells displayed high nuclear/cytoplasmic variance (p < 0.0001). (B) TAZ binding to DVL2 is disrupted in the absence of LATS1. MDA-MB-231 cells stably expressing Flag-TAZ were transfected with siCTL or siLATS1. Endog- enous DVL2 was immunoprecipitated from cell lysates with an anti-DVL2 Fab (see Figure S6), and associated TAZ was analyzed by immunoblotting. (C) Abrogation of LATS expression enhances Wnt3A-dependent DVL2 phosphorylation. MDA-MB-231 cells were transfected with siCTL or siLATS1 and treated with control or Wnt3A-conditioned media for 4 hr. Cell lysates were then analyzed by immunoblotting with the indicated antibodies.

Article Snippet: Immunoprecipitation (IP) and immunoblotting (IB) were carried out with the following antibodies: Flag (Sigma #F3165; 1 mg/IP, 1:2000 IB); T7 (Novagen #69522-3; 1 mg/IP, 1:10,000 IB); HA (Roche #11867431001; 1:1000 IB); TAZ (BD Biosciences #560235; 3 mg/IP, 1:1000 IB); TAZ (Novus #NB600-220; 1:1000 IB); CK13 (BD Biosciences #610445; 1:2000 IB); b-Catenin (BD Biosci- Deve ences #610153; 1:1000 IB); Actin (Sigma # A2066; 1:2000 IB); DVL2 (Cell Signaling Technology #3224; 1:1000 IB); LATS1 (Bethyl #A300-478A; 1:1000 IB); MST1 (BD biosciences #77120; 1:1000 IB); MST2 (Novus #NB10079800; 1:1000 IB).

Techniques: Activity Assay, Inhibition, Expressing, Transfection, Control, Microscopy, Staining, Binding Assay, Stable Transfection, Immunoprecipitation, Western Blot, Phospho-proteomics

Journal: Journal of immunology research

Article Title: Expression and clinical significance of YAP, TAZ, and AREG in hepatocellular carcinoma.

doi: 10.1155/2014/261365

Figure Lengend Snippet: Figure 1: Immunohistochemically stained tissues from HCC patients and adjacent nontumor tissues. Expression of YAP in HCC tissues (a) and adjacent nontumor tissues (b); expression of TAZ in HCC tissues (c) and adjacent nontumor tissues (d); expression of AREG in HCC tissues (e) and adjacent nontumor tissues (f). Representative images were taken under a microscope (×400). These results indicate the clinical significance of YAP, TAZ, and AREG overexpression in HCC tissues.

Article Snippet: Sectionswere then separately incubatedwith the primary antibodies directed against YAP, TAZ, and AREG at 4∘C overnight, detected using biotinylated secondary antibodies (Zhongshan Golden bridge Biotechnology Ltd. Co., China) according to themanufacturer’s recommendations.The staining of the sectionswas performed using theHRP-streptavidin conjugates for YAP, TAZ, and AREG.

Techniques: Staining, Expressing, Microscopy, Over Expression

Journal: Oncotarget

Article Title: Hypoxia-inducible factor 1 mediates TAZ expression and nuclear localization to induce the breast cancer stem cell phenotype.

doi: 10.18632/oncotarget.2997

Figure Lengend Snippet: Figure 1: The WWTR1 gene encoding TAZ is a HIF-1 target gene. (A) Reverse transcription and quantitative real-time PCR (RT-qPCR) were performed to quantify TAZ mRNA levels in 5 breast cell lines following exposure to 20% or 1% O2 for 24 h. For each sample, the expression of TAZ mRNA was quantified relative to 18S rRNA and then normalized to lane 1 (mean ± SEM; n = 3). **P < 0.01, ***P < 0.001 versus MCF-10A at 20% O2; ###P < 0.001 versus MCF-10A at 1% O2. (B) Immunoblot assays were performed to analyze TAZ protein expression in breast cell lines following exposure to 20% (N) or 1% (H) O2 for 48 h. (C) TAZ mRNA expression was analyzed by RT-qPCR in the MDA-MB-231 (left), MDA-MB-435 (middle) and MCF-7 (right) non-targeting control (NTC) or empty vector (EV) subclone and subclones expressing shRNA targeting HIF-1α (sh1α) or HIF-2α (sh2α), which were exposed to 20% or 1% O2 for 24 h. Data were normalized to lane 1 in each bar graph (mean ± SEM; n = 3). ** P < 0.01 versus NTC or EV at 20% O2; ## P < 0.01 versus NTC or EV at 1% O2. (D) Immunoblot assays were performed to analyze HIF-1α, HIF-2α, TAZ, and actin protein expression using lysates prepared from MDA-MB-231 subclones exposed to 20% or 1% O2 for 48 h. (E) The nucleotide sequence (non-coding strand) of a hypoxia response element (HRE; 5’-GCGTG-3’ HIF-1 binding site and 5’-CACA-3’ accessory sequence are shown in red) within intron 2 of the WWTR1 gene, located 26 kb from the transcription start site, is shown. Exons and intron are not drawn to scale. (F) HCC-1954 cells were exposed to 20% or 1% O2 for 16 h and chromatin immunoprecipitation (ChIP) assays were performed using IgG or antibodies against HIF-1α (upper panel), HIF-1β (middle panel), or HIF-2α (lower panel). Primers flanking the HRE were used for qPCR and results were normalized to lane 1 (mean ± SEM; n = 3). *P < 0.05 versus 20% O2 (Student’s t test). (G) The WWTR1 HRE containing a wild type (WT: 5’-GCGTG-3’) or mutant (MUT; 5’-GAAAG-3’) HIF-1 binding site was inserted into pGL2-Promoter (encoding firefly luciferase) and co-transfected with pSV-Renilla (encoding Renilla luciferase) into HCC-1954 cells, which were incubated at 20% or 1% O2 for 24 h. The firefly:Renilla luciferase ratio (Luciferase activity) was normalized to lane 1 (mean ± SEM; n = 3). ***P < 0.001 versus WT at 20% O2; ###P < 0.001 versus WT at 1% O2 (Student’s t test).

Article Snippet: Immunofluorescence MDA-MB-231 cells plated on Lab-Tek II chamber slides (Thermo Fisher Scientific) were fixed with 4% paraformaldehyde (Sigma), permeabilized with 0.1% Triton X-100 (Fisher) for 5 min, blocked with PBS supplemented with 10% BSA for 20 min, and stained with anti-TAZ antibody (Novus Biologicals) overnight at 4°C, followed by incubation with FITC-conjugated goat anti-rabbit IgG (Novus Biologicals).

Techniques: Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Western Blot, Control, Plasmid Preparation, shRNA, Sequencing, Binding Assay, Chromatin Immunoprecipitation, Mutagenesis, Luciferase, Transfection, Incubation, Activity Assay

Journal: Oncotarget

Article Title: Hypoxia-inducible factor 1 mediates TAZ expression and nuclear localization to induce the breast cancer stem cell phenotype.

doi: 10.18632/oncotarget.2997

Figure Lengend Snippet: Figure 3: Analysis of TAZ subcellular localization. (A) MDA-MB-231 subclones (NTC, sh1α, and sh2α) were exposed to 20% or 1% O2 for 48 h and stained with anti-TAZ antibody (green), Alexa Fluor 568-conjugated phalloidin to detect cytosolic F-actin (red), and DAPI to detect nuclear DNA (blue). (B-C) Image analysis was performed to determine the cytoplasmic (B) or nuclear (C) TAZ fluorescence intensity per cell normalized to lane 1 (mean ± SEM; n = 50 cells). *P < 0.05, **P < 0.01, ***P < 0.001 versus NTC at 20% O2; ###P < 0.001 versus NTC at 1% O2 (two-way ANOVA with Bonferroni post-test). (D) Immunoblot assays of TAZ, histone H3, and α-tubulin protein in cytosolic (C) and nuclear (N) lysates prepared from MDA-MB-231 subclones (NTC, sh1α, sh2α) exposed to 20% or 1% O2 for 48 h.

Article Snippet: Immunofluorescence MDA-MB-231 cells plated on Lab-Tek II chamber slides (Thermo Fisher Scientific) were fixed with 4% paraformaldehyde (Sigma), permeabilized with 0.1% Triton X-100 (Fisher) for 5 min, blocked with PBS supplemented with 10% BSA for 20 min, and stained with anti-TAZ antibody (Novus Biologicals) overnight at 4°C, followed by incubation with FITC-conjugated goat anti-rabbit IgG (Novus Biologicals).

Techniques: Staining, Fluorescence, Western Blot

Journal: Oncotarget

Article Title: Hypoxia-inducible factor 1 mediates TAZ expression and nuclear localization to induce the breast cancer stem cell phenotype.

doi: 10.18632/oncotarget.2997

Figure Lengend Snippet: Figure 5: SIAH1 is a HIF-1 target gene product that regulates TAZ nuclear localization. (A) SIAH1 mRNA levels were analyzed by RT-qPCR in MDA-MB-231 subclones (NTC, sh1α, sh2α), which were exposed to 20% or 1% O2 for 24 h. The data were normalized to lane 1 (mean ± SEM; n = 3). *P < 0.05 versus NTC at 20% O2; #P < 0.05 versus NTC at 1% O2. (B) Immunoblot assays were performed using antibodies against HIF-1α, HIF-2α, SIAH1, and actin with WCLs prepared from MDA-MB-231 subclones (NTC, sh1α, sh2α) exposed to 20% or 1% O2 for 48 h. (C) The nucleotide sequence of a candidate HRE (two copies of HIF-1 binding site 5’-GCGTG-3’ are shown in red) within intron 1 of the human SIAH1 gene, located 785 bp 3’ to the transcription start site, is shown. Exons and intron are not drawn to scale. (D) MCF-7 cells were exposed to 20% or 1% O2 for 16 h and ChIP assays were performed using IgG or antibodies against HIF-1α, HIF-2α or HIF-1β. Primers flanking the HRE were used for qPCR and results were normalized to lane 1 (mean ± SEM; n = 3). **P < 0.01 versus 20% O2 (Student’s t test). (E) The SIAH1 HRE containing wild type (WT: 5’-GCGTGAACGGCGTG-3’) or mutant (MUT; 5’-GAAAGAACGGAAAG-3’) HIF-1 binding sites was inserted into pGL2-Promoter (encoding firefly luciferase) and co-transfected with pSV-Renilla (encoding Renilla luciferase) into MCF-7 cells, which were incubated at 20% or 1% O2 for 24 h. The firefly:Renilla luciferase ratio (Luciferase activity) was normalized to lane 1 (mean ± SEM; n = 3). ***P < 0.001 versus WT at 20% O2; ###P < 0.001 versus WT at 1% O2 (Student’s t test). (F-G) NTC or shSI-1 subclones were exposed to 20% or 1% O2, subjected to immunofluorescent assays, and image analysis was performed to determine the nuclear (F) or cytoplasmic (G) TAZ fluorescence intensity per cell normalized to lane 1 (mean ± SEM; n = 50 cells). ***P < 0.001 versus NTC at 20% O2; ###P < 0.001 versus NTC at 1% O2 (two-way ANOVA with Bonferroni post-test). (H) Left panel: Immunoblot assays of TAZ, histone H3, and α-tubulin protein were performed using cytosolic (C) and nuclear (N) lysates prepared from MDA-MB-231 subclones (NTC, shSI1-1, shSI1-2) that were exposed to 20% or 1% O2 for 48 h. Right panel: Immunoblot assays were performed using antibodies against SIAH1 and actin with WCLs prepared from MDA-MB-231 NTC and SIAH1 knockdown subclones.

Article Snippet: Immunofluorescence MDA-MB-231 cells plated on Lab-Tek II chamber slides (Thermo Fisher Scientific) were fixed with 4% paraformaldehyde (Sigma), permeabilized with 0.1% Triton X-100 (Fisher) for 5 min, blocked with PBS supplemented with 10% BSA for 20 min, and stained with anti-TAZ antibody (Novus Biologicals) overnight at 4°C, followed by incubation with FITC-conjugated goat anti-rabbit IgG (Novus Biologicals).

Techniques: Quantitative RT-PCR, Western Blot, Sequencing, Binding Assay, Mutagenesis, Luciferase, Transfection, Incubation, Activity Assay, Fluorescence, Knockdown

Journal: bioRxiv

Article Title: YAP1 Depletion Enhances TAZ and its Complexation with TEAD4 and AP-1 Heterodimer C-JUN/FOSB to Promote Gastric Cancer Progression and Metastases

doi: 10.1101/2025.11.13.683933

Figure Lengend Snippet: A . Increased YAP1 or TAZ in GC tumor tissues compared to normal tissues ( http://gepia.cancer-pku.cn ). B . Both YAP1 and TAZ are significantly correlated (R=0.42; P value=0). C & D . Expression of YAP1 or TAZ is significantly higher in GC tumor tissues than normal and significantly positively correlated (R=0.56, P =0.004) in GSE33335 cohort (** P <0.01; **** P <0.001); E . Expression of YAP1 or TAZ was determined using immunohistochemistry in 390 cases of GC specimen in both intestinal GC and diffuse GC. F . The correlation of YAP1 and TAZ in GC tissues was further confirmed in an individual GC cohort (GSE237876) (R=0.34, P =0.034). G. co-localization of YAP1 and TAZ in two representative PDXs GC tissues was determined by co-immunofluorescent staining of YAP1, TAZ and DAPI (scale bar: 25μm).

Article Snippet: Sections were incubated with primary antibodies: human YAP1 antibody (Cell signaling, cat#14074; 1:100) and human TAZ antibody (Novus, cat#NBP1-85067; 1:100) followed by biotinylated secondary antibodies, and streptavidin HRP kit (Vector Laboratories, PK-6100).

Techniques: Expressing, Immunohistochemistry, Staining

Journal: bioRxiv

Article Title: YAP1 Depletion Enhances TAZ and its Complexation with TEAD4 and AP-1 Heterodimer C-JUN/FOSB to Promote Gastric Cancer Progression and Metastases

doi: 10.1101/2025.11.13.683933

Figure Lengend Snippet: YAP1 and TAZ are highly expressed in GC tumor tissues and PDXs. A. Increased expression of YAP1 or TAZ (WWTR1) in GC tumor tissues compared to normal tissues from a public GC data set (GSE146996). B. Both YAP1 and TAZ are correlated (R=0.19) in the same dataset. C. Expression of YAP1 and TAZ in our own TMA of 390 GC Cases TAZ was statistically analyzed (p=0.001); D. co-expression of YAP1/EpCAM or TAZ/EpCAM was determined by co-IF in two representative PDXs; E. mRNA expression of YAP1 or TAZ was determined in tumor tissues of 18 PDXs from gastroesophageal cancers.

Article Snippet: Sections were incubated with primary antibodies: human YAP1 antibody (Cell signaling, cat#14074; 1:100) and human TAZ antibody (Novus, cat#NBP1-85067; 1:100) followed by biotinylated secondary antibodies, and streptavidin HRP kit (Vector Laboratories, PK-6100).

Techniques: Expressing

Journal: bioRxiv

Article Title: YAP1 Depletion Enhances TAZ and its Complexation with TEAD4 and AP-1 Heterodimer C-JUN/FOSB to Promote Gastric Cancer Progression and Metastases

doi: 10.1101/2025.11.13.683933

Figure Lengend Snippet: A . expression of YAP1 and TAZ in tumor cell clusters was shown in dotted plots in 20 GCPM samples by scRNAseq. B. Coexpression of YAP1 and TAZ in 13 tumor cell clusters was shown in tSNE plots in GCPM samples by scRNAseq C . Expression of YAP1 and TAZ as determined by dual-immunofluorescent staining (co-IF) in representative GCPM samples. Scale bar: 10μm. D . High correlation of YAP1 and TAZ in two metastatic GC cohorts (GSE237876 and GSE289037). E. Expression of YAP1 was significantly increased in peritoneal metastasis compared to primary tumors from a GC cohort (GSE237876).

Article Snippet: Sections were incubated with primary antibodies: human YAP1 antibody (Cell signaling, cat#14074; 1:100) and human TAZ antibody (Novus, cat#NBP1-85067; 1:100) followed by biotinylated secondary antibodies, and streptavidin HRP kit (Vector Laboratories, PK-6100).

Techniques: Expressing, Staining

Journal: bioRxiv

Article Title: YAP1 Depletion Enhances TAZ and its Complexation with TEAD4 and AP-1 Heterodimer C-JUN/FOSB to Promote Gastric Cancer Progression and Metastases

doi: 10.1101/2025.11.13.683933

Figure Lengend Snippet: Co-expression of YAP1 and TAZ in representative GCPM samples. A. coexpression of YAP1 or TAZ in nuclear of two representative GCPM specimen was determined by co-IF staining; B. Coexpression of YAP1 with epithelial tumor marker EpCAM or TAZ or stromal marker vimentin and M2 macrophage marker CD163 in GA0518 GCPM cases; C. TAZ expression was determined in GC with peritoneal metastases compared to GC without peritoneal metastases from a public GC dataset GSE289037.

Article Snippet: Sections were incubated with primary antibodies: human YAP1 antibody (Cell signaling, cat#14074; 1:100) and human TAZ antibody (Novus, cat#NBP1-85067; 1:100) followed by biotinylated secondary antibodies, and streptavidin HRP kit (Vector Laboratories, PK-6100).

Techniques: Expressing, Staining, Marker

Journal: bioRxiv

Article Title: YAP1 Depletion Enhances TAZ and its Complexation with TEAD4 and AP-1 Heterodimer C-JUN/FOSB to Promote Gastric Cancer Progression and Metastases

doi: 10.1101/2025.11.13.683933

Figure Lengend Snippet: A . scRNAseq showed the expression of TEAD1, TEAD2, TEAD3 and TEAD4 by dot plots from 20 GCPM specimens; B . Association of TEAD1-TEAD4 expression with GC patients’ survival in more than 600 advanced GC patients respectively from the TCGA database ( kmplot.com ). C . Representative GCPM cases stained by dual-immunofluorescence of YAP1 with TEAD1-TEAD4 respectively. D . YAP1 interacts with TAZ and TEAD1-TEAD4 in AGS and GA0518 GC cells as shown by co-immunoprecipitation pulldown using anti-YAP1 antibody. E . TAZ interacts with YAP1 and TEAD1-TEAD4 as shown by co-immunoprecipitation pulldown using anti-TAZ antibody in AGS and GA0518 GC cells. F . TEAD transcriptional activity was determined as previously by transient cotransfection of 5X UAS-luciferase reporter and Gal4-TEAD4 with either YAP1cDNA or TAZ cDNA or both in GA0518, GA0804 and AGS cells for 48 hours. *p<0.05, **p<0.01; ***p<0.001.

Article Snippet: Sections were incubated with primary antibodies: human YAP1 antibody (Cell signaling, cat#14074; 1:100) and human TAZ antibody (Novus, cat#NBP1-85067; 1:100) followed by biotinylated secondary antibodies, and streptavidin HRP kit (Vector Laboratories, PK-6100).

Techniques: Expressing, Staining, Immunofluorescence, Immunoprecipitation, Activity Assay, Cotransfection, Luciferase

Journal: bioRxiv

Article Title: YAP1 Depletion Enhances TAZ and its Complexation with TEAD4 and AP-1 Heterodimer C-JUN/FOSB to Promote Gastric Cancer Progression and Metastases

doi: 10.1101/2025.11.13.683933

Figure Lengend Snippet: TAZ ASO suppresses TAZ expression, transcription, TAZ/TEAD transcriptional activity and reduces tumor cell invasion in vitro . A&B. TAZ protein and mRNA levels were decreased by TAZ ASO as determined by Western blot and q-PCR in both GA0518 and GA0804 cells; *p<0.05, **p<0.01; C . TAZ/TEAD transcriptional activity was determined as previously by transient cotransfection of 5X UAS-luciferase reporter and Gal4-TEAD4 with either TAZ cDNA or YAP1cDNA in GA0518 and GA0804 for 48 hours. *p<0.05, **p<0.01; ****p<0.001. D . Luciferase activity of YAP1/TAZ/TEAD was determined after cotransfection of 5X UAS-luciferase reporter and Gal4-TEAD4 with both TAZ cDNA and YAP1cDNA and then treated with YAP1 ASO or TAZ ASO in GA0518 cells; ****p<0.001; E . Luciferase activity of TAZ/TEAD was determined after cotransfection of 5X UAS-luciferase reporter and Gal4-TEAD4 with TAZ cDNA overexpression as done previously. ***p<0.001; F . TAZ ASO strongly suppressed tumor cell invasion capacity in a dose-dependent manner in both GA0518 (left) and GA0804 (right) cells. **p<0.01; ***p<0.001.

Article Snippet: Sections were incubated with primary antibodies: human YAP1 antibody (Cell signaling, cat#14074; 1:100) and human TAZ antibody (Novus, cat#NBP1-85067; 1:100) followed by biotinylated secondary antibodies, and streptavidin HRP kit (Vector Laboratories, PK-6100).

Techniques: Expressing, Activity Assay, In Vitro, Western Blot, Cotransfection, Luciferase, Over Expression

Journal: bioRxiv

Article Title: YAP1 Depletion Enhances TAZ and its Complexation with TEAD4 and AP-1 Heterodimer C-JUN/FOSB to Promote Gastric Cancer Progression and Metastases

doi: 10.1101/2025.11.13.683933

Figure Lengend Snippet: TAZ ASO specifically suppress TAZ expression and inhibit GA0518 cell colony formation. A. Expression of YAP1 and TAZ was determined in GA0518 cells by western blot after treatment of TAZ ASO in dosage as indicated; B&C. Demonstration of colony formation (B) and quantification (C) in GA0518 cells treated with YAP1 ASO, TAZ ASO or a reported YAP1/TEAD inhibitor CA3 at the dosage indicated; *p<0.05, **p<0.01; ***p<0.001.

Article Snippet: Sections were incubated with primary antibodies: human YAP1 antibody (Cell signaling, cat#14074; 1:100) and human TAZ antibody (Novus, cat#NBP1-85067; 1:100) followed by biotinylated secondary antibodies, and streptavidin HRP kit (Vector Laboratories, PK-6100).

Techniques: Expressing, Western Blot

Journal: bioRxiv

Article Title: YAP1 Depletion Enhances TAZ and its Complexation with TEAD4 and AP-1 Heterodimer C-JUN/FOSB to Promote Gastric Cancer Progression and Metastases

doi: 10.1101/2025.11.13.683933

Figure Lengend Snippet: A. TAZ level increased upon YAP1 KO by Western blot (left) and q-PCR (right) in GA0518 cells; B. YAP1 ASO decreased YAP1 but increased TAZ protein (left) and mRNA (right) levels in both GA0518 (above) and GA0804 (below) cells by Western blot and q-PCR respectively; C . YAP1 KO dramatically increased TAZ binding to TEAD4 and the AP1 heterodimer of C-JUN and FOSB as determined by co-IP in GA0518 cells; D . Similarly, inhibition of YAP1 by ASO increased TAZ binding to TEAD4 and the AP1 heterodimer of C-JUN and FOSB as determined by co-IP in GA0518 cells. E . Co-IF staining of TAZ and TEAD4 in four representative GCPM specimen. Scale bar: 25μm. F . Expression of YAP1, TEAD4, c-JUN and FOS-B was determined by co-IF using their specific antibody respectively in GA0518 YAP1 KO clones compared to GA0518 Control cells. Scale bar: 20μm.

Article Snippet: Sections were incubated with primary antibodies: human YAP1 antibody (Cell signaling, cat#14074; 1:100) and human TAZ antibody (Novus, cat#NBP1-85067; 1:100) followed by biotinylated secondary antibodies, and streptavidin HRP kit (Vector Laboratories, PK-6100).

Techniques: Western Blot, Binding Assay, Co-Immunoprecipitation Assay, Inhibition, Staining, Expressing, Clone Assay, Control

Journal: bioRxiv

Article Title: YAP1 Depletion Enhances TAZ and its Complexation with TEAD4 and AP-1 Heterodimer C-JUN/FOSB to Promote Gastric Cancer Progression and Metastases

doi: 10.1101/2025.11.13.683933

Figure Lengend Snippet: A-D. YAP1 and TAZ protein and mRNA levels were determined by western blot and q-PCR upon treatment of YAP1 ASO or TAZ ASO or their combination in both GA0518 and GA0804 cells. *p<0.05, **p<0.01; ***p<0.001. E. Invasion capacity was determined by western blot and q-PCR upon treatment of YAP1 ASO or TAZ ASO or their combination in both GA0518 and GA0804 cells. **p<0.01; ***p<0.001; Scale bar: 25μm; F . Colony formation was determined in GA0804 cells upon treatment of YAP1 ASO or TAZ ASO or their combination. **p<0.01; ***p<0.001.

Article Snippet: Sections were incubated with primary antibodies: human YAP1 antibody (Cell signaling, cat#14074; 1:100) and human TAZ antibody (Novus, cat#NBP1-85067; 1:100) followed by biotinylated secondary antibodies, and streptavidin HRP kit (Vector Laboratories, PK-6100).

Techniques: Western Blot

Journal: bioRxiv

Article Title: YAP1 Depletion Enhances TAZ and its Complexation with TEAD4 and AP-1 Heterodimer C-JUN/FOSB to Promote Gastric Cancer Progression and Metastases

doi: 10.1101/2025.11.13.683933

Figure Lengend Snippet: ASO co-targeting of YAP and TAZ significantly attenuated tumor growth in PDX with greater antitumor effects when combined with anti-PD1 immunotherapy in the KP-Luc syngeneic model. A-B. Combined ASO inhibition of YAP1 and TAZ suppressed tumor weights and volumes in the GA0518 PDX model; YAP1 ASO or TAZ ASO: 50mg/kg, 5 times a week for three weeks. C . Expression of YAP1, TAZ and KI67 as determined by IF staining or immunohistochemistry in YAP1, TAZ, or combination ASO-treated PDX tumors; Scale bar: 20 μm; D. Combination of YAP1 ASO and anti-PD1 antibody had greater antitumor effects than YAP1 ASO alone or the control group; *p<0.05, ***p<0.001; E . Expressions of YAP1, KI67 and SOX9 were decreased by the YAP1 ASO/anti-PD1 combination treatment, while CD8 infiltration was also increased by the combination treatment over either alone. Scale bar: 20 μm; F . Working model and the rationale for co-targeting YAP1 and TAZ in advanced GCPM patients.

Article Snippet: Sections were incubated with primary antibodies: human YAP1 antibody (Cell signaling, cat#14074; 1:100) and human TAZ antibody (Novus, cat#NBP1-85067; 1:100) followed by biotinylated secondary antibodies, and streptavidin HRP kit (Vector Laboratories, PK-6100).

Techniques: Inhibition, Expressing, Staining, Immunohistochemistry, Control

Journal: bioRxiv

Article Title: YAP1 Depletion Enhances TAZ and its Complexation with TEAD4 and AP-1 Heterodimer C-JUN/FOSB to Promote Gastric Cancer Progression and Metastases

doi: 10.1101/2025.11.13.683933

Figure Lengend Snippet: ASO co-targeting of YAP and TAZ significantly attenuated tumor growth in additional PDX. A. Combined ASO inhibition of YAP1 and TAZ suppressed tumor weights in a GC PDX model; YAP1 ASO or TAZ ASO: 50mg/kg, the combination of YAP1 ASO (25mg/kg) and TAZ ASO (25mg/kg); 5 times a week for three weeks. B. Actually tumor sizes in different treatment groups were shown at the end of the experiment. C. co-expression of TAZ/vimentin and YAP1/EpCAM as determined by IF staining in YAP1 ASO, TAZ ASO, or combination ASO-treated PDX tumors; Scale bar: 20 μm;

Article Snippet: Sections were incubated with primary antibodies: human YAP1 antibody (Cell signaling, cat#14074; 1:100) and human TAZ antibody (Novus, cat#NBP1-85067; 1:100) followed by biotinylated secondary antibodies, and streptavidin HRP kit (Vector Laboratories, PK-6100).

Techniques: Inhibition, Expressing, Staining