Journal: Nature communications

Article Title: Tumour suppressor TRIM33 targets nuclear β-catenin degradation

doi: 10.1038/ncomms7156

Figure Lengend Snippet: a, Co-IP of endogenous TRIM33 with β-catenin in U87/EGFRvIII cells ( left panel ) and DLD-1 cells ( right panel ). TRIM33 was immunoprecipitated, and the amount of TRIM33 bound to β-catenin was determined using an immunoblot with an anti-β-catenin antibody. b, Interaction of endogenous β-catenin ( top panel ) and endogenous TRIM33 ( bottom panel ) was induced upon Wnt3a treatment. IP was performed with whole-cell lysates of HEK 293T cells pretreated with 100 ng/ml Wnt3a. Whole-cell lysates were probed for input. The amounts of TRIM33 and β-catenin immunoprecipitated were normalized with IgG. c, The C-terminal domain of β-catenin mediates the interaction of this protein with TRIM33. Top panel, Schematic illustration of β-catenin. delC and delN constructs lack the C terminus and N terminus of the protein, respectively. Bottom panel, β-Catenin deletion mutants were co-expressed with FLAG-TRIM33 in 293T cells in the presence of MG132. The cells were subjected to IP with a FLAG antibody followed by immunoblotting (IB) with FLAG and Myc antibodies. Whole-cell lysates were directly subjected to IB using FLAG or Myc antibody as input.

Article Snippet: The following antibodies were from Bethyl Laboratories: TRIM33 (A301-060A; 1:1,000 IB, 2 μg IP), TRIM33 (IHC-00216; 1:100 IHC).

Techniques: Co-Immunoprecipitation Assay, Immunoprecipitation, Western Blot, Construct

Journal: Nature communications

Article Title: Tumour suppressor TRIM33 targets nuclear β-catenin degradation

doi: 10.1038/ncomms7156

Figure Lengend Snippet: a , Left panels, U87/EGFRvIII cells or SW480 cells were transfected with an empty vector or FLAG-tagged TRIM33 expression vector. Right panels, U87 cells or DLD-1 cells were transduced using TRIM33 shRNA. Cell lysates were collected at 48 h post-transfection and subjected to immunoblot analyses using the indicated antibodies. b, TRIM33 destabilized nuclear β-catenin protein expression. Left panel, Western blot of nuclear β-catenin in control-shRNA or TRIM33-shRNA U87 cells as indicated. Cells were harvested at the indicated times following inhibition of new protein synthesis by cyclohexamide (CHX). Right panel , Quantitation of nuclear β-catenin normalized to the loading control and expressed relative to 0 h. c , Cellular levels of Cyclin D1 and Axin2 in U87 cells depleted of endogenous TRIM33 were analysed by immunoblot analyses. Columns #1 and #2 indicate two independent shRNAs targeting TRIM33 . d , Cell fractions were prepared from control-shRNA or TRIM33-shRNA U87 cells. Lamin B, tubulin, and Na-K-ATPase served as loading controls and cell fraction markers. e , U87/EGFRvIII cells were transfected with or without FLAG-tagged TRIM33 treated with MG132 as indicated. Nuclear cell lysates were subjected to SDS-PAGE and immunoblot analysis using anti-β-catenin or anti-FLAG antibodies.

Article Snippet: The following antibodies were from Bethyl Laboratories: TRIM33 (A301-060A; 1:1,000 IB, 2 μg IP), TRIM33 (IHC-00216; 1:100 IHC).

Techniques: Transfection, Plasmid Preparation, Expressing, shRNA, Western Blot, Control, Inhibition, Quantitation Assay, SDS Page

Journal: Nature communications

Article Title: Tumour suppressor TRIM33 targets nuclear β-catenin degradation

doi: 10.1038/ncomms7156

Figure Lengend Snippet: a, GSK-3β −/− MEFs were transfected with the constructs indicated; 48 h later the cells were harvested and the nuclear extracts analysed by IB. b–d, HEK 293T cells were co-transfected with the indicated constructs; 48 h later the cells were harvested and the nuclear extracts analysed by IB. e , Top panel, Diagram of TRIM33 protein domains. The N-terminal domain (TRIM) consists of a RING-finger domain, two B-box domains (B) and a coiled-coil domain (CC); the C-terminal domain consists of plant homeodomain (PHD)/bromodomain (Br) regions. Both domains are coupled by a middle region. Bottom panel, FLAG-TRIM33 and β-catenin-Myc were co-expressed in HEK 293T cells in the presence of MG132. FLAG-TRIM33 protein was immunoprecipitated using an anti-FLAG antibody. β-Catenin–bound TRIM33 protein was detected using an immunoblot with an anti-Myc antibody. Whole-cell lysates were directly subjected to IB using anti-Myc antibody as input. f, U87/EGFRvIII cells were transfected with the indicated plasmids, the nuclear extracts were prepared, and the β-catenin level was examined. Lamin B was used as a loading control for nuclear fractions. g , HEK 293T cells were transfected with the indicated plasmids. Transfection efficiency was normalized by co-transfection with the pRL-TK plasmid. Luciferase activity was measured 36 h after transfection by the dual luciferase assay. Luciferase activity is shown as relative luciferase activity (RLA) compared to that in cells transfected with the control vector. h , HEK 293T cells were transfected with the indicated plasmids. Luciferase activity was measured 36 h after transfection. i , HEK 293T cells transfected with or without TRIM33 were transfected with TOP-FLASH or FOP-FLASH, which was followed by Wnt3a treatment for 4 h. j , U87 cells transfected with or without TRIM33 were transfected with TOP-FLASH or FOP-FLASH, which was followed by EGF treatment for 10 h. In g , h , i , and j , each error bar indicates the variation between the means of 3 independent experiments.

Article Snippet: The following antibodies were from Bethyl Laboratories: TRIM33 (A301-060A; 1:1,000 IB, 2 μg IP), TRIM33 (IHC-00216; 1:100 IHC).

Techniques: Transfection, Construct, Immunoprecipitation, Western Blot, Control, Cotransfection, Plasmid Preparation, Luciferase, Activity Assay

Journal: Nature communications

Article Title: Tumour suppressor TRIM33 targets nuclear β-catenin degradation

doi: 10.1038/ncomms7156

Figure Lengend Snippet: a, HEK 293T cells with or without TRIM33 depletion by siRNA were transfected as indicated. Cells were treated with MG132 (10 μM for 4 h) prior to lysis and then subjected to anti-β-catenin IP followed by anti-HA with immunoblot analysis. b, HEK 293T cells with or without TRIM33 depletion by shRNA were transfected as indicated. Experiments were performed as described for a. c , Nuclear extracts of U87/EGFRvIII cells pretreated with MG132 (10 μM for 4 h) were immunoprecipitated. ΔRING, TRIM33 lacking the RING domain; CAmut, TRIM33 with point mutations (C125A/C128A) in the RING domain. d , In vivo ubiquitination assays performed in HEK 293T cells transiently transfected with HA-tagged K63-only ubiquitin or K48-only ubiquitin. e, TRIM33 ubiquitylation of β-catenin in vitro . Purified β-catenin was subjected to in vitro ubiquitylation by TRIM33 in the absence or presence of active PKCδ. β-Catenin was detected with anti-GST antibody. f & g, U87 cells with or without TRIM33 depletion were transfected with β-TrCP siRNA or control siRNA. Cells were treated for 8 h with 100 ng/ml Wnt-3a (Wnt-on) (f) or with 100 ng DKK1 (Wnt-off) (g). Experiments were performed as described for a.

Article Snippet: The following antibodies were from Bethyl Laboratories: TRIM33 (A301-060A; 1:1,000 IB, 2 μg IP), TRIM33 (IHC-00216; 1:100 IHC).

Techniques: Transfection, Lysis, Western Blot, shRNA, Immunoprecipitation, In Vivo, Ubiquitin Proteomics, In Vitro, Purification, Control

Journal: Nature communications

Article Title: Tumour suppressor TRIM33 targets nuclear β-catenin degradation

doi: 10.1038/ncomms7156

Figure Lengend Snippet: a, HEK 293T cells were transfected with the indicated plasmids. Luciferase activity was measured 36 h after transfection. Each error bar indicates the variation between the means of 3 independent experiments. b, HEK 293T cells were transfected with FLAG-tagged TRIM33 and the indicated Myc-tagged β-catenin mutants. Whole-cell lysates were immunoprecipitated with anti-FLAG antibody, and immunocomplexes were probed with antibodies against the indicated proteins. CAmut, TRIM33 with point mutations (C125A/C128A) in the RING domain. c, HEK 293T cells were transfected with FLAG-tagged TRIM33 and the indicated Myc-tagged β-catenin mutants. Experiments were performed as described for b. d, Alignment of the amino acid regions containing the TRIM33 binding motif in β-catenin orthologues. e, β-Catenin S715A mutants had a longer protein half-life. Left panel, HEK 293T cells were transfected with the indicated mutants. Cells were incubated with cyclohexamide (CHX) for the indicated times, collected, and analysed by IB as indicated. Right panel, the amount of β-catenin (WT or mutants) is represented relative to the amount at time 0. f, HEK 293T cells were transfected with FLAG-TRIM33 alone or in combination with β-catenin WT –Myc or β-catenin–Myc serine substitution mutants. The transfected cells were treated with MG132 4 h prior to harvest, which was followed by IP with anti-Myc antibody. Cell lysates and the immunoprecipitates were analysed by IB with the indicated antibodies.

Article Snippet: The following antibodies were from Bethyl Laboratories: TRIM33 (A301-060A; 1:1,000 IB, 2 μg IP), TRIM33 (IHC-00216; 1:100 IHC).

Techniques: Transfection, Luciferase, Activity Assay, Immunoprecipitation, Binding Assay, Incubation

Journal: Nature communications

Article Title: Tumour suppressor TRIM33 targets nuclear β-catenin degradation

doi: 10.1038/ncomms7156

Figure Lengend Snippet: a, HEK 293T cells transiently transfected with or without TRIM33 were transfected with the indicated constructs. Cells were pretreated with Bis-I (2 mM) or Go6976 (2 mM). Each error bar indicates the variation between the means of 3 independent experiments. b, U87/EGFRvIII cells were transiently transfected with constitutively active (+) or kinase-dead (−) PKC mutants. Nuclear cell lysates were collected at 48 h post-transfection and subjected to immunoblot analyses using the indicated antibodies. c, U87 cells with or without TRIM33 depletion were transfected with siRNAs targeting PKCδ or control siRNA, incubated with cyclohexamide (CHX) for the indicated times, and collected and analysed. Top panel , IB results. Bottom panel , Quantitation of nuclear β-catenin was normalized to the loading control and expressed relative to 0 h. d, PKCδ silencing inhibited TRIM33–β-catenin interaction. U87/EGFRvIII cells were transfected with either a siRNA targeting PKCδ or control siRNA; 48 h post-transfection, cells were collected and whole-cell lysates were immunoprecipitated and immunoblotted as indicated. e, PKCδ stimulated the binding of TRIM33 to β-catenin. U87 cells were transiently transfected with constitutively active (+) or kinase-dead (−) PKCδ. The transfected cells were treated with MG132 4 h prior to harvest. The experiment was performed as described in d. f & g, Localization of endogenous PKCδ in HEK 293T (f) and U87 (g) cells. Nuclei were visualized with DAPI (blue). Scale bars: 20 μm. h, In vitro kinase assays were performed with purified active PKCδ and purified β-catenin proteins and analysed by autoradiography. i, U87 cells were treated with Wnt3a (100 ng/ml) with or without MG132 for the indicated times. IB was performed with nuclear lysates of the cells with indicated antibody. j, U87 cells transfected with either PKCδ siRNA targeting or control siRNA were treated with Wnt3a (100 ng/ml) for 8 h. IB was performed with nuclear lysates of the cells with anti-pS715-β-catenin antibody.

Article Snippet: The following antibodies were from Bethyl Laboratories: TRIM33 (A301-060A; 1:1,000 IB, 2 μg IP), TRIM33 (IHC-00216; 1:100 IHC).

Techniques: Transfection, Construct, Western Blot, Control, Incubation, Quantitation Assay, Immunoprecipitation, Binding Assay, In Vitro, Purification, Autoradiography

Journal: Nature communications

Article Title: Tumour suppressor TRIM33 targets nuclear β-catenin degradation

doi: 10.1038/ncomms7156

Figure Lengend Snippet: a, U87 cells with or without depleted TRIM33 and shβ-catenin were intracranially injected into athymic nude mice. After 2 weeks, the mice were sacrificed and tumour growth was examined. Left panel, top, Haematoxylin and eosin (H&E)-stained coronal brain sections show representative tumour xenografts. Bottom, nuclear β-catenin levels were detected by IB analyses in these established stable cells. Right panel, Tumor volumes were measured by using length ( a ) and width ( b ) and were calculated using the equation: V = ab 2 /2. Data represent results of 5 mice per group of two independent experiments. Error bars ± SD. Significance was determined by Mann–Whitney U -test. b, U87/EGFRvIII cells with or without TRIM33 and β-catenin (WT or mutant) overexpression were intracranially injected into athymic nude mice. After 2 weeks, the mice were sacrificed and tumour growth was examined. Left panel, top, H&E-stained coronal brain sections show representative tumour xenografts. Bottom, nuclear β-catenin levels were detected by IB analyses in these established stable cells. Right panel, Tumor volumes were measured using the equation: V = ab 2 /2. Data represent results of 5 mice per group of two independent experiments. Error bars z U -test. c, TRIM33-depleted U87 cells stably expressing β-catenin knockdown and their proliferation were analysed by MTT assay. Data are presented as mean ± SD from 3 independent experiments. d, Soft agar assays for TRIM33-depleted U87 cells stably expressing β-catenin knockdown. Data are presented as mean ± SD from 3 independent experiments. e, TRIM33 overexpression U87/EGFRvIII cells stably expressing β-catenin (WT or mutant). Proliferation was analysed by MTT assay. f, Soft agar assay for TRIM33-overexpressing U87/EGFRvIII cells stably expressing β-catenin (WT or mutant). g, Brain tumours produced by U87/EGFRvIII cells in nude mice were processed and sectioned for immunostaining with specific antibodies against TRIM33 and active β-catenin. H&E staining revealed histopathologic features of normal mouse brain tissue and a U87/EGFRvIII tumour. Scale bars: 200 μm.

Article Snippet: The following antibodies were from Bethyl Laboratories: TRIM33 (A301-060A; 1:1,000 IB, 2 μg IP), TRIM33 (IHC-00216; 1:100 IHC).

Techniques: Injection, Staining, MANN-WHITNEY, Mutagenesis, Over Expression, Stable Transfection, Expressing, Knockdown, MTT Assay, Soft Agar Assay, Produced, Immunostaining

Journal: Nature communications

Article Title: Tumour suppressor TRIM33 targets nuclear β-catenin degradation

doi: 10.1038/ncomms7156

Figure Lengend Snippet: a, IHC staining with anti-TRIM33 and anti-active β-catenin antibodies was carried out on 40 human GBM specimens. Photographs of two representative tumours are shown. Scale bars: 200 μm. b, Semiquantitative scoring was carried out ( r =−0.528, P <0.001, Pearson correlation coefficient) with all 40 tumours. Note that some of the dots on the graphs represent more than one specimen (i.e., some scores overlapped). c, Comparison the expression level of TRIM33 in tumours with that of the adjacent normal brain tissues in 28 specimens. Boxes indicate interquartile range. Bars from each box extend to the largest and smallest observations. P <0.001 (Student’s t -test). d, Model for TRIM33-mediated degradation of nuclear β-catenin. Left, in cells with a level of TRIM33 which is comparable to normal cells, Wnt treatment induces the canonical Wnt/β-catenin signaling pathway activation and stabilizes β-catenin. Stabilized β-catenin is translocated to the nucleus forms a complex with TCF/LEF-1 transcription factors which controls the expression of many genes such as Cyclin D1 . Our novel findings demonstrate that prolonged Wnt stimulation can lead to activation of PKCδ; then, phosphorylation of β-catenin at Ser715 by activated PKCδ facilitates the interaction between TRIM33 and β-catenin, which induce β-catenin degradation and hence shut down Wnt signaling. Thus, the TRIM33-dependent β-catenin degradation serves as a negative feedback loop to shut down Wnt signaling upon prolonged activation of the Wnt signaling. Right , in many tumour cells such as GBM cells, the level of TRIM33 is down-regulated, which would result in inactivating this negative feedback loop and hence constitutive activation of the Wnt signaling pathway to promote tumour cell proliferation and facilitate tumorigenesis.

Article Snippet: The following antibodies were from Bethyl Laboratories: TRIM33 (A301-060A; 1:1,000 IB, 2 μg IP), TRIM33 (IHC-00216; 1:100 IHC).

Techniques: Immunohistochemistry, Comparison, Expressing, Activation Assay, Phospho-proteomics

Journal: The Journal of Biological Chemistry

Article Title: Downregulation of PIK3IP1/TrIP on T cells is controlled by TCR signal strength, PKC, and metalloprotease-mediated cleavage

doi: 10.1016/j.jbc.2024.107930

Figure Lengend Snippet: Development and validation of novel monoclonal antibodies to the ecto domain of TrIP. A , cloning strategy and development of the ecto-TrIP Fc-fusion protein used for immunization and antibody development. B , SDS-PAGE of the ectoTrIP-Ig fusion protein under reducing and nonreducing conditions. C , flow cytometry data showing the binding of each anti-TrIP monoclonal antibody to a Flag-tagged WT murine TrIP construct expressed in HEK293T cells. D , flow cytometry data showing staining of the clones on HEK293T cells transfected with a plasmid encoding Flag-tagged human TrIP. E , flow cytometry data showing the staining of each of the anti-TrIP mAbs on HEK293T cells transfected with a Flag-tagged mTrIP construct lacking the extracellular kringle domain (ΔKringle). HEK, human embryonic kidney; mAb, monoclonal antibody; mTrIP, murine TrIP; TrIP, transmembrane inhibitor of PI3K.

Article Snippet: Monoclonal antibodies to the ecto domain of murine TrIP were developed in conjunction with a commercial vendor (Rockland, Inc).

Techniques: Biomarker Discovery, Bioprocessing, Cloning, SDS Page, Flow Cytometry, Binding Assay, Construct, Staining, Clone Assay, Transfection, Plasmid Preparation

Journal: BioMed Research International

Article Title: TRIM33 Overexpression Inhibits the Progression of Clear Cell Renal Cell Carcinoma In Vivo and In Vitro

doi: 10.1155/2020/8409239

Figure Lengend Snippet: TRIM33 overexpression can inhibit migration, invasion, and EMT in ccRCC; here, the potential molecular mechanisms are explored. (a and b) Wound healing assay. (c–e) Transwell assay, including migration and invasion experiments. (f–h) Western blot assay for TRIM33, E-cadherin, N-cadherin, vimentin, β -catenin, cyclin D1, c-myc, and β -actin. (i–k) Western blot assay after incubation with a Wnt signaling pathway agonist for β -catenin, cyclin D1, c-myc, and β -actin. ∗∗ P < 0.01; ∗∗∗ P < 0.001; ∗∗∗∗ P < 0.0001; ns represents no statistical significance.

Article Snippet: The antibodies being used included rabbit anti-TRIM33 (Abcam, 47062 and 84455), anti- β -actin (Abcam, 8227), anti-E-cadherin (Abcam, 15148), anti-N-cadherin (Abcam, 18203), anti-vimentin (Abcam, 92547), anti- β -catenin (Abcam, 16051), anti-cyclin D1 (Abcam, ab40754), and anti-c-myc (Abcam, 32072) antibodies.

Techniques: Over Expression, Migration, Wound Healing Assay, Transwell Assay, Western Blot, Incubation

Journal: BioMed Research International

Article Title: TRIM33 Overexpression Inhibits the Progression of Clear Cell Renal Cell Carcinoma In Vivo and In Vitro

doi: 10.1155/2020/8409239

Figure Lengend Snippet: TRIM33 overexpression can inhibit tumor growth in vivo. (a) Pictures of nude mice and tumors. (b) Scatter plot of tumor weight between the OV-TRIM33 group and the OV-NC group. P < 0.0001. (c) Tumor volume measured every 3 days after the second week. (d, e) Immunohistochemistry staining for TRIM33, E-cadherin, N-cadherin, and vimentin. A histogram is used to display semiquantitative analysis results. ∗∗∗ P < 0.001; ∗∗∗∗ P < 0.0001.

Article Snippet: The antibodies being used included rabbit anti-TRIM33 (Abcam, 47062 and 84455), anti- β -actin (Abcam, 8227), anti-E-cadherin (Abcam, 15148), anti-N-cadherin (Abcam, 18203), anti-vimentin (Abcam, 92547), anti- β -catenin (Abcam, 16051), anti-cyclin D1 (Abcam, ab40754), and anti-c-myc (Abcam, 32072) antibodies.

Techniques: Over Expression, In Vivo, Immunohistochemistry, Staining