Journal: Science Advances

Article Title: TEAD4 is a master regulator of high-risk nasopharyngeal carcinoma

doi: 10.1126/sciadv.add0960

Figure Lengend Snippet: ( A ) The workflow for the inference of MR proteins of NPC at high risk of progression. ( B ) Heatmap showing the average expression of the 13 candidate MRs in high- and low-aggressiveness groups of malignant cells from cohort 3 (GSE150430 dataset). Division of high- and low-aggressiveness groups according to the median value of scores for a cell cycling signature derived from our previous study of 5397 malignant cells profiled by scRNA-seq. P values were calculated using Wilcoxon rank sum test. ( C ) Box plots depicting the expression of YAP , TAZ , and TEAD1 to TEAD4 from the Hippo signaling pathway in NPC ( n = 31) versus normal ( n = 10) tissue samples from the GSE12452 dataset, showing a significant difference only for TEAD4 . The box plot center corresponds to the median, the upper and lower lines of the box correspond to the interquartile range, and the whiskers correspond to the 1.5× interquartile range. P values were calculated using Wilcoxon rank sum test. ( D and E ) Forest plot (D) and Kaplan-Meier curves (E) showing associations of the expression levels of YAP , TAZ , and TEAD1 to TEAD4 with the progression-free survival of 82 patients from the GSE102349 dataset. A significant association was shown only for TEAD4 . The expression of YAP , TAZ , and TEAD1 to TEAD4 was divided into high and low groups according to their median values, respectively. P values in (D) and (E) were calculated using a log-rank test.

Article Snippet: Cell lysates were incubated with anti-YAP (13584-1-AP, Proteintech), anti-TAZ (23306-1-AP, Proteintech), or anti-Myc (2276S, Cell Signaling Technology) antibodies at 4°C overnight.

Techniques: Expressing, Derivative Assay

Journal: Science Advances

Article Title: TEAD4 is a master regulator of high-risk nasopharyngeal carcinoma

doi: 10.1126/sciadv.add0960

Figure Lengend Snippet: ( A ) Lysates from SUNE-1 cells were immunoprecipitated with anti-YAP or anti-TAZ antibodies and subjected to Western blot analysis with anti-TEAD4 and anti-YAP or anti-TAZ antibodies. ( B ) Gel filtration chromatography analysis of SUNE-1 cell lysates. Forty-eight fractions (A1 to A12, B1 to B12, C1 to C12, and D1 to D12) were collected and the A11 to C6 fractions were shown; proteins pooled from two fractions were run on each lane. ( C ) Lysates from SUNE-1 cells transfected with Myc-TEAD4 WT , Myc-TEAD4 Y429H , Myc-TEAD4 K297A , or Myc-TEAD4 W299A were immunoprecipitated with an anti-Myc antibody and subjected to Western blot analysis with the anti-Myc, anti-YAP, or anti-TAZ antibodies. ( D ) Representative images (left) and quantification of migratory cells (right) of Transwell migration assays in TEAD4-knockdown SUNE-1 and HONE-1 cells transfected with plasmids encoding TEAD4 WT or TEAD4 Y429H . Scale bar, 100 μm. ( E ) Representative images (left) and apoptosis rate (right) of cell apoptosis assays in TEAD4-knockdown SUNE-1 cells transfected with plasmids encoding TEAD4 WT or TEAD4 Y429H and exposed to PBS or cisplatin (DDP). ( F ) Western blot analysis of TEAD4, YAP/TAZ, BZW2, p-AKT, and AKT in TEAD4-knockdown SUNE-1 and HONE-1 cells transfected with plasmids encoding TEAD4 WT , TEAD4 Y429H , TEAD4 K297A , or TEAD4 W299A . Data in (D) and (E) are presented as means ± SD; n = 3 independent experiments. P values were calculated using one-way ANOVA. * P < 0.05 and ** P < 0.01.

Article Snippet: Cell lysates were incubated with anti-YAP (13584-1-AP, Proteintech), anti-TAZ (23306-1-AP, Proteintech), or anti-Myc (2276S, Cell Signaling Technology) antibodies at 4°C overnight.

Techniques: Immunoprecipitation, Western Blot, Filtration, Chromatography, Transfection, Migration

Journal: Science Advances

Article Title: TEAD4 is a master regulator of high-risk nasopharyngeal carcinoma

doi: 10.1126/sciadv.add0960

Figure Lengend Snippet: ( A ) Representative images of IHC staining for TEAD4 in NPC tissue samples, in which the expression level was measured using the immunoreactive score (IRS) system. Scale bars, 100 μm. ( B ) Distribution of TEAD4 IRS in our NPC cohort ( n = 219). ( C to E ) Kaplan-Meier curves of disease-free survival (C), distant metastasis-free survival (D), and overall survival (E) according to high and low TEAD4 expression in 219 patients with NPC. Hazard ratios (HRs) were calculated using multivariate analysis with a CPH model (table S5). P values were calculated using a log-rank test. ( F and G ) Kaplan-Meier curves of disease-free survival in patients with NPC from low (F) and high (G) TEAD4 expression groups that were treated with or without cisplatin-based IC. P values were calculated using a log-rank test. ( H ) A treatment-by-covariate (TEAD4 expression * cisplatin-based IC) interaction test with the CPH model on disease-free survival showing that the treatment effect of cisplatin-based IC varied in high versus low TEAD4 expression groups. ( I ) Proposed working model of TEAD4 in NPC. YTHDF2 recognizes WTAP-mediated TEAD4 m 6 A methylation to facilitate its stability and leads to aberrant up-regulation of TEAD4 in NPC. Up-regulated TEAD4 expression further promotes NPC metastasis and chemoresistance by transcriptionally promoting the downstream BZW2, which inhibits PHLPP2 to activate the AKT pathway, independent of YAP/TAZ modulation.

Article Snippet: Cell lysates were incubated with anti-YAP (13584-1-AP, Proteintech), anti-TAZ (23306-1-AP, Proteintech), or anti-Myc (2276S, Cell Signaling Technology) antibodies at 4°C overnight.

Techniques: Immunohistochemistry, Expressing, Methylation

Journal: Frontiers in Oncology

Article Title: Ring finger protein 128 promotes, rather than inhibits, colorectal cancer progression by regulating the Hippo signaling pathway

doi: 10.3389/fonc.2022.1031160

Figure Lengend Snippet: RNF128 inhibits the Hippo pathway in colorectal cancer. (A) RNF128 was correlated with Hippo pathway proteins (LATS, YAP, and TAZ) in CRC according to StarBase. (B) RNF128 was downregulated in LoVo and HCT116 cells transfected with si-NC and si-RNF128, as shown by western blot analysis. ** P <0.001. (C) Western blot analysis was used to measure the protein expression of components of the Hippo pathway (MST, p-MST, LATS, p-LATS, YAP, p-YAP, and TAZ) in LoVo and HCT116 cells with RNF128 knockdown. ** P <0.001.

Article Snippet: The antibodies used were as follows: RNF128 (Abcam 72533, 1:1000), MST (ZEN-BIOSCIENCE 382248, 1:1000; Proteintech 22245-1-AP, 1:1000), p-LATS (Affinity AF8163, 1:1000), YAP (Affinity DF3182, 1:1000), p-YAP (Affinity AF3328, 1:1000), TAZ (Proteintech 23306-1-AP, 1:1000), β-actin (Bimake A5092, 1:2000), IgG (ABclonal AC005), Bcl-2 (Proteintech 60178-1-Ig, 1:1000), HIF-1α (Ploneer in Proteomics PTM-5851, 1:1000), IκBα (Proteintech 10268-1-AP, 1:1000), p-IκBα (Santa Cruz Biotechnology sc-8404, 1:500) and Ub (Proteintech 10201-2-AP, 1:1000).

Techniques: Transfection, Western Blot, Expressing

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Mechanism by Which Tong Xie Yao Fang Heals the Intestinal Mucosa of Rats with Ulcerative Colitis through the Hippo Pathway

doi: 10.1155/2021/5533914

Figure Lengend Snippet: The sequences of the RT-qPCR primers.

Article Snippet: Then, the proteins were separated and transferred to polyvinylidene difluoride membranes, which were probed with anti-YAP1 monoclonal antibody (1 : 1000), anti-P-YAP antibody (1 : 1000), anti-TAZ monoclonal antibody (1 : 1000), anti-LATS1 monoclonal antibody (1 : 1000), anti-Claudin-1 monoclonal antibody (1 : 1000), and anti-Caspase 3 (1 : 1000, Proteintech Group, Inc., Wuhan, China), respectively.

Techniques:

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Mechanism by Which Tong Xie Yao Fang Heals the Intestinal Mucosa of Rats with Ulcerative Colitis through the Hippo Pathway

doi: 10.1155/2021/5533914

Figure Lengend Snippet: In the early stage of inflammation, TXYF inhibited Hippo pathway activity. (a) The expression of YAP1 from colon tissue was detected by immunohistochemistry on the 7th day. The mRNA levels of YAP1 (b), TAZ (c), and LATS1 (d) from colon mucosa were detected by RT-qPCR analysis on the 7th day. The protein expressions of YAP1, TAZ, P-YAP, and LATS1 from colon mucosa were detected by western blot on the 3rd day (e) and the 7th day (f). (g) The relative protein expression of LATS1 and YAP1 from colon tissue of TXYF group in different stages of colon inflammation. GAPDH was served as internal control ( n = 3 in each group). ∧∧ P < 0.01 versus control group; ∗ P < 0.05 and ∗∗ P < 0.01 versus model group; # P < 0.05 and ## P < 0.01 versus sulfasalazine group; @@ P < 0.01 versus the earlier stage.

Article Snippet: Then, the proteins were separated and transferred to polyvinylidene difluoride membranes, which were probed with anti-YAP1 monoclonal antibody (1 : 1000), anti-P-YAP antibody (1 : 1000), anti-TAZ monoclonal antibody (1 : 1000), anti-LATS1 monoclonal antibody (1 : 1000), anti-Claudin-1 monoclonal antibody (1 : 1000), and anti-Caspase 3 (1 : 1000, Proteintech Group, Inc., Wuhan, China), respectively.

Techniques: Activity Assay, Expressing, Immunohistochemistry, Quantitative RT-PCR, Western Blot

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Mechanism by Which Tong Xie Yao Fang Heals the Intestinal Mucosa of Rats with Ulcerative Colitis through the Hippo Pathway

doi: 10.1155/2021/5533914

Figure Lengend Snippet: In the late stage of inflammation, TXYF activated Hippo pathway activity. (a) The expression of YAP1 from colon tissues was detected by immunohistochemistry on the 28th day. The mRNA levels of YAP1 (b), TAZ (c), and LATS1 (d) from colon mucosa were detected by RT-qPCR analysis on the 28th day. The protein expressions of YAP1, TAZ, P-YAP, and LATS1 from colon mucosa were detected by western blot on the 14th day and the 28th day. ∧∧ P < 0.01 versus control group; ∗ P < 0.05 and ∗∗ P < 0.01 versus model group; # P < 0.05 and ## P < 0.01 versus sulfasalazine group.

Article Snippet: Then, the proteins were separated and transferred to polyvinylidene difluoride membranes, which were probed with anti-YAP1 monoclonal antibody (1 : 1000), anti-P-YAP antibody (1 : 1000), anti-TAZ monoclonal antibody (1 : 1000), anti-LATS1 monoclonal antibody (1 : 1000), anti-Claudin-1 monoclonal antibody (1 : 1000), and anti-Caspase 3 (1 : 1000, Proteintech Group, Inc., Wuhan, China), respectively.

Techniques: Activity Assay, Expressing, Immunohistochemistry, Quantitative RT-PCR, Western Blot

Journal: Journal of Cancer

Article Title: MiR-34a-5p Inhibits Proliferation, Migration, Invasion and Epithelial-mesenchymal Transition in Esophageal Squamous Cell Carcinoma by Targeting LEF1 and Inactivation of the Hippo-YAP1/TAZ Signaling Pathway

doi: 10.7150/jca.39861

Figure Lengend Snippet: miR-34a-5p repressed Hippo-YAP1/TAZ signaling pathway. (a) RNA-Seq data showed that significant change was taken place in Hippo signaling pathway after LEF1 was overexpressed. (b,c) YAP1/TAZ expression was downregulated after miR-34a-5p upregulation in Eca109 or TE1 cells compared to the control groups.

Article Snippet: Protein extracts were subjected to SDS-PAGE and analyzed using the following primary antibodies: LEF1 antibody (Abcam, ab137872), YAP1 antibody (Abcam, ab52771), TAZ antibody (Proteintech, 23306-1-AP), E-cadherin antibody(Abcam, ab40772), N-cadherin antibody (Abcam, ab18203) and GADPH antibody (Abcam, ab8245).

Techniques: RNA Sequencing Assay, Expressing

Journal: Frontiers in Pharmacology

Article Title: TAZ Induces Migration of Microglia and Promotes Neurological Recovery After Spinal Cord Injury

doi: 10.3389/fphar.2022.938416

Figure Lengend Snippet: The changes of TAZ after the polarization of microglia and myelin treatment. (A) Western blot was used to detect the changes of TAZ expression in the different microglial phenotypes and myelin treatment ( n = 3 per group), microglia were differentiated into pro-inflammatory M1-like (M1-L), anti-inflammatory M2-like (M2-L), and resting, unstimulated (M0-L) phenotypes. (B) Quantitative analysis of TAZ expression in (A) . GAPDH was used as the loading control. Data were mean ± SEM. * p <0.05 (M0-L vs. M2-L); ** p <0.01 (M1-L vs. M2-L); ** p <0.01 (M0-L vs. Meylin); *** p <0.001 (M1-L vs. Meylin). (C) Representative immunofluorescence images of TAZ (green) in M0-L, M1-L, M2-L microglia and myelin-treated microglia, and the nuclei were stained with DAPI (blue). Yellow arrows indicated that TAZ gathered around the nuclei of microglia. White arrows indicated that TAZ accumulated the nuclei of microglia. Scale bars: low magnification, 100 μM; higher magnification, 20 μM.

Article Snippet: The cells were incubated overnight at 4°C with primary antibodies, the primary antibodies included mouse anti-TAZ (1:100, Proteintech, 66500-1-lg), rabbit anti-Fascin-1 (1:100, Abcam, ab126772).

Techniques: Western Blot, Expressing, Immunofluorescence, Staining

Journal: Frontiers in Pharmacology

Article Title: TAZ Induces Migration of Microglia and Promotes Neurological Recovery After Spinal Cord Injury

doi: 10.3389/fphar.2022.938416

Figure Lengend Snippet: Fascin-1 upregulated TAZ and mediated TAZ into the nuclei of microglia in vitro . (A) Western blot was used to detect the expression levels of Fascin-1 and TAZ after transfection with siFascin-1 (knockdown) and siNC (control) ( n = 3 per group). (B,C) Quantitative analysis of the relative levels of Fascin-1 (B) and TAZ (C) as shown in (A). Protein expression was normalized to GAPDH. Data were mean ± SEM. * p <0.05 (siNC vs. siFascin-1#2) in (B); ** p <0.01 (siNC vs siFascin-1#3) in (B); * p <0.05 (siNC vs. siFascin-1#2 or siFascin-1#3) in (C). The results showed that knockdown siFascin-1#3 was the best effect on transfection. (D) After transfection of siFascin-1 and siNC into microglia in vitro , double immunostaining analysis of TAZ (green) and Fascin-1 (red) in microglia. Scale bars: low magnification, 100 μM; higher magnification, 20 μM. Yellow arrows indicated that TAZ gathered around the nuclei of microglia. (E) Western blot was used to exam the expression levels of Fascin-1 and TAZ after transfection with plasmid Flag-Fascin-1 (overexpression) and Flag (control) ( n = 3 per group). (F,G) Quantitative analysis of the relative levels of Fascin-1 (F) and TAZ (G) as shown in (E). Data were mean ± SEM. ** p <0.01 (Flag vs Flag-Fascin-1) in (F). * p <0.05 (Flag vs. Flag-Fascin-1). (H) Double immunostaining analysis of TAZ (green) and Fascin-1 (red) in microglia after transfection with Flag and Flag-Fascin-1. White arrows indicated that TAZ accumulated the nuclear of microglia. Scale bars: low magnification, 100 μM; higher magnification, 20 μM.

Article Snippet: The cells were incubated overnight at 4°C with primary antibodies, the primary antibodies included mouse anti-TAZ (1:100, Proteintech, 66500-1-lg), rabbit anti-Fascin-1 (1:100, Abcam, ab126772).

Techniques: In Vitro, Western Blot, Expressing, Transfection, Double Immunostaining, Plasmid Preparation, Over Expression

Journal: Frontiers in Pharmacology

Article Title: TAZ Induces Migration of Microglia and Promotes Neurological Recovery After Spinal Cord Injury

doi: 10.3389/fphar.2022.938416

Figure Lengend Snippet: Fascin-1 interacted with TAZ and mediated the entry of TAZ into the nuclei of microglia in vivo . (A) Coimmunoprecipitation (Co-IP) analysis of the interaction between Fascin-1 and TAZ in myelin-treated microglia and 7 days SCI tissues. IP, immunoprecipitation; Input, total cells and tissue lysate; WB, Western blot analysis. IgG was used as a control IP. Pull down of the endogenous Fascin-1 protein complex was performed using mouse monoclonal Fascin-1 antibody. Fascin-1 and TAZ proteins were detected after Western blot the resulting immunoprecipitates using the rabbit polyclonal Fascin-1 antibody, rabbit monoclonal TAZ antibody. (B) Immunofluorescence markers from the sagittal section of the spinal cords showed the spatio-temporal distribution of Fascin-1 (red), TAZ (green), and DAPI (blue) at pre, 7 and 14 days after SCI. White arrows indicated that TAZ accumulated the nuclei of Fascin-1 + microglia. The asterisks indicated the centre of the lesion. Scale bars: low magnification, 100 μM; higher magnification, 20μM.

Article Snippet: The cells were incubated overnight at 4°C with primary antibodies, the primary antibodies included mouse anti-TAZ (1:100, Proteintech, 66500-1-lg), rabbit anti-Fascin-1 (1:100, Abcam, ab126772).

Techniques: In Vivo, Co-Immunoprecipitation Assay, Immunoprecipitation, Western Blot, Immunofluorescence

Journal: Frontiers in Pharmacology

Article Title: TAZ Induces Migration of Microglia and Promotes Neurological Recovery After Spinal Cord Injury

doi: 10.3389/fphar.2022.938416

Figure Lengend Snippet: TAZ was downstream of Fascin-1 for regulating microglial migration. (A) Western blot was used to detect protein expression changes of Fascin-1 and TAZ after different treatment groups, including Flag+siNC, Flag-Fascin-1+siNC, Flag-Fascin-1+siTAZ ( n = 3 per group). (B,C) Quantitative analysis of the relative levels of Fascin-1 (B) and TAZ (C) as shown in (A). The protein expression was normalized to GAPDH. Data were mean ± SEM. * p <0.05 (Flag-Fascin-1+siNC vs Flag+siNC); ** p <0.01 (Flag-Fascin-1+siTAZ vs Flag-Fascin-1+siNC) in (B and C). (D,F) Scratch and Transwell tests were used to detect microglial migration in the above treatment groups ( n = 3 per group). (E) Quantitative analysis of the number of transmembrane cells in (D). Data were mean ± SEM. ** p <0.01 (Flag-Fascin-1+siNC vs. Flag+siNC), ** p <0.01 (Flag-Fascin-1+siTAZ vs. Flag-Fascin-1+siNC). Scale bar: 200 μM. (G) Quantification of blank area ratio in (F). Data were mean ± SEM. **** p <0.0001 (Flag-Fascin-1+siNC vs. Flag+siNC), **** p <0.0001 (Flag-Fascin-1+siTAZ vs. Flag-Fascin-1+siNC). Scale bar: 200 μM.

Article Snippet: The cells were incubated overnight at 4°C with primary antibodies, the primary antibodies included mouse anti-TAZ (1:100, Proteintech, 66500-1-lg), rabbit anti-Fascin-1 (1:100, Abcam, ab126772).

Techniques: Migration, Western Blot, Expressing

Journal: Frontiers in Pharmacology

Article Title: TAZ Induces Migration of Microglia and Promotes Neurological Recovery After Spinal Cord Injury

doi: 10.3389/fphar.2022.938416

Figure Lengend Snippet: TAZ can promote the migration and accumulation of microglia around the lession core to form microglial scar after SCI and Fascin-1-TAZ pathway axis for positively regulating the migration of microglia. In this model, TAZ was downstream of Fascin-1 for regulating microglial migration. TAZ promoted the migration of microglia by Fascin-1 inducing TAZ nuclear accumulation of microglia, which contributed to the migration and accumulation of microglia around the lesion border to form microglial scars and promote functional recovery after SCI.

Article Snippet: The cells were incubated overnight at 4°C with primary antibodies, the primary antibodies included mouse anti-TAZ (1:100, Proteintech, 66500-1-lg), rabbit anti-Fascin-1 (1:100, Abcam, ab126772).

Techniques: Migration, Functional Assay