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anti trim37 (Bethyl)

Name: anti trim37
Brand: Bethyl
Rating: 93 (10 reviews)

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Bethyl anti trim37

RIME analysis reveals AP-2γ is associated with <t>TRIM37</t> in BCa cells. A, Graphical plot showing AP-2γ-associated proteins in MCF-7 cells. The clustering of the graph is based on molecular functions. B, Peptide coverage, number of unique peptides (highlighted in blue) identified in AP-2γ and TRIM37 from the AP-2γ RIME analysis. C, TRIM37 mRNA alteration in different cancer types from TCGA. D, TRIM37 expression in TRIM37 diploid versus TRIM37-amplified tumors (TCGA). E, TRIM37 mRNA levels in normal and different BCa subtypes from different publicly available datasets. The number of patients and p-values are shown as indicated. F, Validation of the AP-2γ-TRIM37 interaction by co-immunoprecipitation. Nuclear AP-2γ protein in MCF-7 (left) or BT474 (right) cells was immunoprecipitated, and TRIM37 was detected by immunoblotting. IgG was used as a negative control.

Anti Trim37, supplied by Bethyl, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 93 stars, based on 1 article reviews

anti trim37 - by Bioz Stars, 2025-02

93/100 stars

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1) Product Images from "TRIM37 Augments AP-2γ Transcriptional Activity and Cellular Localization via K63-linked Ubiquitination to Drive Breast Cancer Progression"

Article Title: TRIM37 Augments AP-2γ Transcriptional Activity and Cellular Localization via K63-linked Ubiquitination to Drive Breast Cancer Progression

Journal: International Journal of Biological Sciences

doi: 10.7150/ijbs.69466

RIME analysis reveals AP-2γ is associated with TRIM37 in BCa cells. A, Graphical plot showing AP-2γ-associated proteins in MCF-7 cells. The clustering of the graph is based on molecular functions. B, Peptide coverage, number of unique peptides (highlighted in blue) identified in AP-2γ and TRIM37 from the AP-2γ RIME analysis. C, TRIM37 mRNA alteration in different cancer types from TCGA. D, TRIM37 expression in TRIM37 diploid versus TRIM37-amplified tumors (TCGA). E, TRIM37 mRNA levels in normal and different BCa subtypes from different publicly available datasets. The number of patients and p-values are shown as indicated. F, Validation of the AP-2γ-TRIM37 interaction by co-immunoprecipitation. Nuclear AP-2γ protein in MCF-7 (left) or BT474 (right) cells was immunoprecipitated, and TRIM37 was detected by immunoblotting. IgG was used as a negative control.

Figure Legend Snippet: RIME analysis reveals AP-2γ is associated with TRIM37 in BCa cells. A, Graphical plot showing AP-2γ-associated proteins in MCF-7 cells. The clustering of the graph is based on molecular functions. B, Peptide coverage, number of unique peptides (highlighted in blue) identified in AP-2γ and TRIM37 from the AP-2γ RIME analysis. C, TRIM37 mRNA alteration in different cancer types from TCGA. D, TRIM37 expression in TRIM37 diploid versus TRIM37-amplified tumors (TCGA). E, TRIM37 mRNA levels in normal and different BCa subtypes from different publicly available datasets. The number of patients and p-values are shown as indicated. F, Validation of the AP-2γ-TRIM37 interaction by co-immunoprecipitation. Nuclear AP-2γ protein in MCF-7 (left) or BT474 (right) cells was immunoprecipitated, and TRIM37 was detected by immunoblotting. IgG was used as a negative control.

Techniques Used: Expressing, Amplification, Immunoprecipitation, Western Blot, Negative Control

TRIM37 binding is co-localized with AP-2γ. A, Venn diagram showing the overlap of AP-2γ and TRIM37 ChIP-seq peaks. B, Graphs showing the average tag density for AP-2γ and TRIM37 at AP-2γ-TRIM37 co-binding regions (top). Heatmaps showing the binding intensity signals for the 5,146 sites ranked from the strongest to the weakest binding sites (below). C, Motif enrichment analysis of TRIM37 binding sites. Significant top enriched motifs in TRIM37 ChIP-seq peaks are shown with corresponding p-values. D, Pie chart illustrating the genomic distribution of TRIM37 binding regions relative to the whole genome. E, Genomic snapshots of AP-2γ and TRIM37 ChIP-seq peaks at the regulatory regions of model genes. Asterisks denote binding sites used for validation in F. F, ChIP-qPCR validation of AP-2γ and TRIM37 co-occupancy at model genes in MCF-7 cells. Data are represented as a percentage of input chromatin immunoprecipitated. Error bars show ± SD from at least 3 independent experiments. * P < 0.05; ** P < 0.01.

Figure Legend Snippet: TRIM37 binding is co-localized with AP-2γ. A, Venn diagram showing the overlap of AP-2γ and TRIM37 ChIP-seq peaks. B, Graphs showing the average tag density for AP-2γ and TRIM37 at AP-2γ-TRIM37 co-binding regions (top). Heatmaps showing the binding intensity signals for the 5,146 sites ranked from the strongest to the weakest binding sites (below). C, Motif enrichment analysis of TRIM37 binding sites. Significant top enriched motifs in TRIM37 ChIP-seq peaks are shown with corresponding p-values. D, Pie chart illustrating the genomic distribution of TRIM37 binding regions relative to the whole genome. E, Genomic snapshots of AP-2γ and TRIM37 ChIP-seq peaks at the regulatory regions of model genes. Asterisks denote binding sites used for validation in F. F, ChIP-qPCR validation of AP-2γ and TRIM37 co-occupancy at model genes in MCF-7 cells. Data are represented as a percentage of input chromatin immunoprecipitated. Error bars show ± SD from at least 3 independent experiments. * P < 0.05; ** P < 0.01.

Techniques Used: Binding Assay, ChIP-sequencing, Immunoprecipitation

TRIM37 is required for efficient AP-2γ dependent transcription. A, Effect of AP-2γ and TRIM37 knockdown on the expression of AP-2γ-regulated genes. MCF-7 cells (upper panel) and BT474 cells (lower panel) were transfected with siAP-2γ, siTRIM37, or siNC. Gene expression was measured by RT-qPCR and normalized against GAPDH. Error bars show ± SD from at least 3 independent experiments. * P < 0.05; ** P < 0.01. B, Venn diagram showing AP-2γ and TRIM37 coregulated genes. MCF-7 cells treated with siNC, siAP-2γ, or siTRIM37 were profiled by RNA-seq. A total of 2,880 genes are coregulated by AP-2γ and TRIM37 (AP-2γ FC > 1.5, TRIM37 FC > 1.2). C, Heatmap showing RNA-seq gene expression analysis of MCF-7 cells treated with siAP-2γ, siTRIM37, or siNC. A total of 968 downregulated genes (blue) and 1,581 upregulated genes (red) (AP-2γ FC > 1.5, TRIM37 FC > 1.2) were identified. D, GO analysis of downregulated genes (coactivated by TRIM37 and AP-2γ) (AP-2γ FC > 1.5, TRIM37 FC > 1.2).

Figure Legend Snippet: TRIM37 is required for efficient AP-2γ dependent transcription. A, Effect of AP-2γ and TRIM37 knockdown on the expression of AP-2γ-regulated genes. MCF-7 cells (upper panel) and BT474 cells (lower panel) were transfected with siAP-2γ, siTRIM37, or siNC. Gene expression was measured by RT-qPCR and normalized against GAPDH. Error bars show ± SD from at least 3 independent experiments. * P < 0.05; ** P < 0.01. B, Venn diagram showing AP-2γ and TRIM37 coregulated genes. MCF-7 cells treated with siNC, siAP-2γ, or siTRIM37 were profiled by RNA-seq. A total of 2,880 genes are coregulated by AP-2γ and TRIM37 (AP-2γ FC > 1.5, TRIM37 FC > 1.2). C, Heatmap showing RNA-seq gene expression analysis of MCF-7 cells treated with siAP-2γ, siTRIM37, or siNC. A total of 968 downregulated genes (blue) and 1,581 upregulated genes (red) (AP-2γ FC > 1.5, TRIM37 FC > 1.2) were identified. D, GO analysis of downregulated genes (coactivated by TRIM37 and AP-2γ) (AP-2γ FC > 1.5, TRIM37 FC > 1.2).

Techniques Used: Expressing, Transfection, Quantitative RT-PCR, RNA Sequencing Assay

TRIM37 affects AP-2γ binding to chromatin. A, Graph showing the average tag density (top) and heatmap (bottom) of AP-2γ at AP-2γ and TRIM37 co-bound regions with and without siTRIM37 treatment. B, Genomic snapshots of AP-2γ ChIP-seq peaks surrounding model genes with and without siTRIM37 treatment. C, ChIP-qPCR validation of TRIM37 knockdown on AP-2γ binding sites at model genes in MCF-7 cells. Data are represented as a percentage of input chromatin immunoprecipitated. Error bars show ± SD from at least 3 independent experiments. * P < 0.05; ** P < 0.01.

Figure Legend Snippet: TRIM37 affects AP-2γ binding to chromatin. A, Graph showing the average tag density (top) and heatmap (bottom) of AP-2γ at AP-2γ and TRIM37 co-bound regions with and without siTRIM37 treatment. B, Genomic snapshots of AP-2γ ChIP-seq peaks surrounding model genes with and without siTRIM37 treatment. C, ChIP-qPCR validation of TRIM37 knockdown on AP-2γ binding sites at model genes in MCF-7 cells. Data are represented as a percentage of input chromatin immunoprecipitated. Error bars show ± SD from at least 3 independent experiments. * P < 0.05; ** P < 0.01.

Techniques Used: Binding Assay, ChIP-sequencing, Immunoprecipitation

$TRIM37 affects AP-2γ nuclear translocation via K63 ubiquitination. A, MCF-7 cells transfected with siNC or siTRIM37 were treated with MG132 before lysis and then subjected to anti-AP-2γ IP followed by immunoblot analysis with anti-HA. B, HEK293T cells were transfected with constructs expressing AP-2γ and wild-type TRIM37 or TRIM37[C18R] mutant as indicated. Samples were processed as described in A. The asterisks indicate the mono-ubiquitinated forms of AP-2γ. C, MCF-7 cells were transfected with siNC or siTRIM37. Cell lysis was collected at 72 h post-transfection and subjected to immunoblot analysis using the indicated antibodies. D, MCF-7 cells were transfected with constructs expressing TRIM37 or TRIM37[C18R]. Samples were processed as described in C. E, Cell fractions were prepared from MCF-7 cells treated with siNC or siTRIM37. GAPDH and histone H3 served as loading controls and cell fraction markers. F, Quantitation of cytoplasmic (left) or nuclear (right) AP-2γ normalized to the loading control. Error bars show ± SD from at least 3 independent experiments. * P < 0.05; ** P < 0.01. G, In vivo ubiquitination assays were performed in HEK293T cells. Cells were transiently transfected with HA-tagged K63-only ubiquitin or K48-only ubiquitin. The asterisks indicate the mono-ubiquitinated forms of AP-2γ.$
Figure Legend Snippet: TRIM37 affects AP-2γ nuclear translocation via K63 ubiquitination. A, MCF-7 cells transfected with siNC or siTRIM37 were treated with MG132 before lysis and then subjected to anti-AP-2γ IP followed by immunoblot analysis with anti-HA. B, HEK293T cells were transfected with constructs expressing AP-2γ and wild-type TRIM37 or TRIM37[C18R] mutant as indicated. Samples were processed as described in A. The asterisks indicate the mono-ubiquitinated forms of AP-2γ. C, MCF-7 cells were transfected with siNC or siTRIM37. Cell lysis was collected at 72 h post-transfection and subjected to immunoblot analysis using the indicated antibodies. D, MCF-7 cells were transfected with constructs expressing TRIM37 or TRIM37[C18R]. Samples were processed as described in C. E, Cell fractions were prepared from MCF-7 cells treated with siNC or siTRIM37. GAPDH and histone H3 served as loading controls and cell fraction markers. F, Quantitation of cytoplasmic (left) or nuclear (right) AP-2γ normalized to the loading control. Error bars show ± SD from at least 3 independent experiments. * P < 0.05; ** P < 0.01. G, In vivo ubiquitination assays were performed in HEK293T cells. Cells were transiently transfected with HA-tagged K63-only ubiquitin or K48-only ubiquitin. The asterisks indicate the mono-ubiquitinated forms of AP-2γ.

Techniques Used: Translocation Assay, Transfection, Lysis, Western Blot, Construct, Expressing, Mutagenesis, Quantitation Assay, In Vivo

TRIM37 is essential for BCa growth. A and B, MTT assays were performed on (A) MCF-7 and (B) BT474 cells transfected with siNC or siTRIM37. Error bars represent ± SD from 3 independent experiments. * P < 0.05; ** P < 0.01. C and D, Cell apoptosis analyses by flow cytometry of (C) MCF-7 and (D) BT474 transfected with siNC or siTRIM37. Error bars show ± SD from at least 3 independent experiments. * P < 0.05; ** P < 0.01. E, Cell cycle analysis of MCF-7 cells transfected with siNC or siTRIM37 were detected by flow cytometry. Error bars show ± SD from at least 3 independent experiments. * P < 0.05; ** P < 0.01. F-H, Kaplan-Meier plots showing the recurrence-free survival of BCa patients for TRIM37 (F), AP-2γ (G), TRIM37, and AP-2γ (H) expression in the METABRIC dataset. BCa patients were stratified into high (red) and low (blue) groups based on the median expression of the target gene. Significance was calculated using a log-rank test ( P < 0.05).

Figure Legend Snippet: TRIM37 is essential for BCa growth. A and B, MTT assays were performed on (A) MCF-7 and (B) BT474 cells transfected with siNC or siTRIM37. Error bars represent ± SD from 3 independent experiments. * P < 0.05; ** P < 0.01. C and D, Cell apoptosis analyses by flow cytometry of (C) MCF-7 and (D) BT474 transfected with siNC or siTRIM37. Error bars show ± SD from at least 3 independent experiments. * P < 0.05; ** P < 0.01. E, Cell cycle analysis of MCF-7 cells transfected with siNC or siTRIM37 were detected by flow cytometry. Error bars show ± SD from at least 3 independent experiments. * P < 0.05; ** P < 0.01. F-H, Kaplan-Meier plots showing the recurrence-free survival of BCa patients for TRIM37 (F), AP-2γ (G), TRIM37, and AP-2γ (H) expression in the METABRIC dataset. BCa patients were stratified into high (red) and low (blue) groups based on the median expression of the target gene. Significance was calculated using a log-rank test ( P < 0.05).

Techniques Used: Transfection, Flow Cytometry, Cell Cycle Assay, Expressing

Image Search Results

Journal: International Journal of Biological Sciences

Article Title: TRIM37 Augments AP-2γ Transcriptional Activity and Cellular Localization via K63-linked Ubiquitination to Drive Breast Cancer Progression

doi: 10.7150/ijbs.69466

Figure Lengend Snippet: RIME analysis reveals AP-2γ is associated with TRIM37 in BCa cells. A, Graphical plot showing AP-2γ-associated proteins in MCF-7 cells. The clustering of the graph is based on molecular functions. B, Peptide coverage, number of unique peptides (highlighted in blue) identified in AP-2γ and TRIM37 from the AP-2γ RIME analysis. C, TRIM37 mRNA alteration in different cancer types from TCGA. D, TRIM37 expression in TRIM37 diploid versus TRIM37-amplified tumors (TCGA). E, TRIM37 mRNA levels in normal and different BCa subtypes from different publicly available datasets. The number of patients and p-values are shown as indicated. F, Validation of the AP-2γ-TRIM37 interaction by co-immunoprecipitation. Nuclear AP-2γ protein in MCF-7 (left) or BT474 (right) cells was immunoprecipitated, and TRIM37 was detected by immunoblotting. IgG was used as a negative control.

Article Snippet: The following antibodies were used for RIME, ChIP, Co-IP and immunoblotting: anti-AP-2γ (sc-12762x, SCBT), anti-AP-2γ (sc-8977, SCBT), anti-TRIM37 (A301-174A, Bethyl Lab), anti-HA (sc-7392, SCBT), normal mouse IgG (sc-2025, SCBT), normal rabbit IgG (sc-2027, SCBT), and anti-GAPDH (sc-47724, SCBT).

Techniques: Expressing, Amplification, Immunoprecipitation, Western Blot, Negative Control

Journal: International Journal of Biological Sciences

Article Title: TRIM37 Augments AP-2γ Transcriptional Activity and Cellular Localization via K63-linked Ubiquitination to Drive Breast Cancer Progression

doi: 10.7150/ijbs.69466

Figure Lengend Snippet: TRIM37 binding is co-localized with AP-2γ. A, Venn diagram showing the overlap of AP-2γ and TRIM37 ChIP-seq peaks. B, Graphs showing the average tag density for AP-2γ and TRIM37 at AP-2γ-TRIM37 co-binding regions (top). Heatmaps showing the binding intensity signals for the 5,146 sites ranked from the strongest to the weakest binding sites (below). C, Motif enrichment analysis of TRIM37 binding sites. Significant top enriched motifs in TRIM37 ChIP-seq peaks are shown with corresponding p-values. D, Pie chart illustrating the genomic distribution of TRIM37 binding regions relative to the whole genome. E, Genomic snapshots of AP-2γ and TRIM37 ChIP-seq peaks at the regulatory regions of model genes. Asterisks denote binding sites used for validation in F. F, ChIP-qPCR validation of AP-2γ and TRIM37 co-occupancy at model genes in MCF-7 cells. Data are represented as a percentage of input chromatin immunoprecipitated. Error bars show ± SD from at least 3 independent experiments. * P < 0.05; ** P < 0.01.

Techniques: Binding Assay, ChIP-sequencing, Immunoprecipitation

Journal: International Journal of Biological Sciences

Article Title: TRIM37 Augments AP-2γ Transcriptional Activity and Cellular Localization via K63-linked Ubiquitination to Drive Breast Cancer Progression

doi: 10.7150/ijbs.69466

Figure Lengend Snippet: TRIM37 is required for efficient AP-2γ dependent transcription. A, Effect of AP-2γ and TRIM37 knockdown on the expression of AP-2γ-regulated genes. MCF-7 cells (upper panel) and BT474 cells (lower panel) were transfected with siAP-2γ, siTRIM37, or siNC. Gene expression was measured by RT-qPCR and normalized against GAPDH. Error bars show ± SD from at least 3 independent experiments. * P < 0.05; ** P < 0.01. B, Venn diagram showing AP-2γ and TRIM37 coregulated genes. MCF-7 cells treated with siNC, siAP-2γ, or siTRIM37 were profiled by RNA-seq. A total of 2,880 genes are coregulated by AP-2γ and TRIM37 (AP-2γ FC > 1.5, TRIM37 FC > 1.2). C, Heatmap showing RNA-seq gene expression analysis of MCF-7 cells treated with siAP-2γ, siTRIM37, or siNC. A total of 968 downregulated genes (blue) and 1,581 upregulated genes (red) (AP-2γ FC > 1.5, TRIM37 FC > 1.2) were identified. D, GO analysis of downregulated genes (coactivated by TRIM37 and AP-2γ) (AP-2γ FC > 1.5, TRIM37 FC > 1.2).

Techniques: Expressing, Transfection, Quantitative RT-PCR, RNA Sequencing Assay

Journal: International Journal of Biological Sciences

Article Title: TRIM37 Augments AP-2γ Transcriptional Activity and Cellular Localization via K63-linked Ubiquitination to Drive Breast Cancer Progression

doi: 10.7150/ijbs.69466

Figure Lengend Snippet: TRIM37 affects AP-2γ binding to chromatin. A, Graph showing the average tag density (top) and heatmap (bottom) of AP-2γ at AP-2γ and TRIM37 co-bound regions with and without siTRIM37 treatment. B, Genomic snapshots of AP-2γ ChIP-seq peaks surrounding model genes with and without siTRIM37 treatment. C, ChIP-qPCR validation of TRIM37 knockdown on AP-2γ binding sites at model genes in MCF-7 cells. Data are represented as a percentage of input chromatin immunoprecipitated. Error bars show ± SD from at least 3 independent experiments. * P < 0.05; ** P < 0.01.

Techniques: Binding Assay, ChIP-sequencing, Immunoprecipitation

Journal: International Journal of Biological Sciences

Article Title: TRIM37 Augments AP-2γ Transcriptional Activity and Cellular Localization via K63-linked Ubiquitination to Drive Breast Cancer Progression

doi: 10.7150/ijbs.69466

Figure Lengend Snippet: TRIM37 affects AP-2γ nuclear translocation via K63 ubiquitination. A, MCF-7 cells transfected with siNC or siTRIM37 were treated with MG132 before lysis and then subjected to anti-AP-2γ IP followed by immunoblot analysis with anti-HA. B, HEK293T cells were transfected with constructs expressing AP-2γ and wild-type TRIM37 or TRIM37[C18R] mutant as indicated. Samples were processed as described in A. The asterisks indicate the mono-ubiquitinated forms of AP-2γ. C, MCF-7 cells were transfected with siNC or siTRIM37. Cell lysis was collected at 72 h post-transfection and subjected to immunoblot analysis using the indicated antibodies. D, MCF-7 cells were transfected with constructs expressing TRIM37 or TRIM37[C18R]. Samples were processed as described in C. E, Cell fractions were prepared from MCF-7 cells treated with siNC or siTRIM37. GAPDH and histone H3 served as loading controls and cell fraction markers. F, Quantitation of cytoplasmic (left) or nuclear (right) AP-2γ normalized to the loading control. Error bars show ± SD from at least 3 independent experiments. * P < 0.05; ** P < 0.01. G, In vivo ubiquitination assays were performed in HEK293T cells. Cells were transiently transfected with HA-tagged K63-only ubiquitin or K48-only ubiquitin. The asterisks indicate the mono-ubiquitinated forms of AP-2γ.

Techniques: Translocation Assay, Transfection, Lysis, Western Blot, Construct, Expressing, Mutagenesis, Quantitation Assay, In Vivo

Journal: International Journal of Biological Sciences

Article Title: TRIM37 Augments AP-2γ Transcriptional Activity and Cellular Localization via K63-linked Ubiquitination to Drive Breast Cancer Progression

doi: 10.7150/ijbs.69466

Figure Lengend Snippet: TRIM37 is essential for BCa growth. A and B, MTT assays were performed on (A) MCF-7 and (B) BT474 cells transfected with siNC or siTRIM37. Error bars represent ± SD from 3 independent experiments. * P < 0.05; ** P < 0.01. C and D, Cell apoptosis analyses by flow cytometry of (C) MCF-7 and (D) BT474 transfected with siNC or siTRIM37. Error bars show ± SD from at least 3 independent experiments. * P < 0.05; ** P < 0.01. E, Cell cycle analysis of MCF-7 cells transfected with siNC or siTRIM37 were detected by flow cytometry. Error bars show ± SD from at least 3 independent experiments. * P < 0.05; ** P < 0.01. F-H, Kaplan-Meier plots showing the recurrence-free survival of BCa patients for TRIM37 (F), AP-2γ (G), TRIM37, and AP-2γ (H) expression in the METABRIC dataset. BCa patients were stratified into high (red) and low (blue) groups based on the median expression of the target gene. Significance was calculated using a log-rank test ( P < 0.05).

Techniques: Transfection, Flow Cytometry, Cell Cycle Assay, Expressing

Journal: eLife

Article Title: Global cellular response to chemical perturbation of PLK4 activity and abnormal centrosome number

doi: 10.7554/eLife.73944

Figure Lengend Snippet:

Article Snippet: Antibody , TRIM37 (rabbit, polyclonal) , Bethyl Laboratories , A301-174A , Western blot (1:250) IF (1:250).

Techniques: Clone Assay, Western Blot, Recombinant, Plasmid Preparation, Sequencing, Luciferase, Immunoprecipitation, Software, Transfection, Mutagenesis

( A ) RPE-1 Cas9 cells were stably infected with virus directing the expression of one of two sgRNAs against TRIM37 or empty vector. Selected cells were treated with DMSO, or 200 or 500 nM centrinone B for 4 days, fixed and stained for CEP135 and foci counted. Means and standard deviation shown (n=3, N≥169). ( B ) Cells from ( A ) were also processed for Western blotting using the indicated antibodies. FL – full length. p60 – p60 fragment. Ponc.S indicates total protein. ( C ) Asynchronous RPE-1 cells were fixed and stained with the indicated antibodies. Pairwise merged images are shown (bottom). ( D ) Asynchronus RPE-1 Cas9 cells were fixed and stained for TRIM37, PCNA, and CEP120. The number of TRIM37-positive centrosomes was manually determined for each cell cycle stage. Means from each replicate are shown as open circles. Resulting mean and standard deviation shown (n=3, N≥96). p -value from one-way ANOVA. ( E ) and ( F ) RPE-1 Cas9 cells were fixed and stained for the indicated antibodies. Examples of different cell cycles stages and TRIM37 localizations are shown in ( E ) and ( F ), respectively. Arrowhead in ( E ) indicates TRIM37 preference for one of two centrosomes. M/D: mother/daughter, M: mother, D: daughter. ( G ) Quantification of cells shown in ( E ) and ( F ). Individual data points shown as open circles. REsulting mean and standard deviation show (n=3, N = ≥94). Significant p -values (< 0.05) from a pairwise t-test between G1 and S/G2 populations indicated. ( H ) RPE-1 TRIM37 -/- cells stably expressing FB-TRIM37 were fixed, stained with the indicated antibody, and imaged using 3D-SIM. Two representative images are shown. ( I ) RPE-1 TRIM37 -/- cells stably expressing the indicated construct (FB = FLAG BirA) were pre-extracted, fixed, and stained for the indicated protein. ( J ) Centrosomal TRIM37 signal from cells in ( I ) was quantified. Means from each replicate are shown as open circles. Resulting mean and standard deviation shown (n=3, N≥84). Significant p -values (< 0.05) from Dunnett post hoc test using ‘WT’ as control after one-way ANOVA shown. Note that the results from ( I ) and ( J ) and those in are from the same experiment therefore ‘FLAG-BirA’ and ‘WT’ are duplicated in these panels. See . Figure 3—source data 1. Source data for .

Journal: eLife

Article Title: Global cellular response to chemical perturbation of PLK4 activity and abnormal centrosome number

doi: 10.7554/eLife.73944

Figure Lengend Snippet: ( A ) RPE-1 Cas9 cells were stably infected with virus directing the expression of one of two sgRNAs against TRIM37 or empty vector. Selected cells were treated with DMSO, or 200 or 500 nM centrinone B for 4 days, fixed and stained for CEP135 and foci counted. Means and standard deviation shown (n=3, N≥169). ( B ) Cells from ( A ) were also processed for Western blotting using the indicated antibodies. FL – full length. p60 – p60 fragment. Ponc.S indicates total protein. ( C ) Asynchronous RPE-1 cells were fixed and stained with the indicated antibodies. Pairwise merged images are shown (bottom). ( D ) Asynchronus RPE-1 Cas9 cells were fixed and stained for TRIM37, PCNA, and CEP120. The number of TRIM37-positive centrosomes was manually determined for each cell cycle stage. Means from each replicate are shown as open circles. Resulting mean and standard deviation shown (n=3, N≥96). p -value from one-way ANOVA. ( E ) and ( F ) RPE-1 Cas9 cells were fixed and stained for the indicated antibodies. Examples of different cell cycles stages and TRIM37 localizations are shown in ( E ) and ( F ), respectively. Arrowhead in ( E ) indicates TRIM37 preference for one of two centrosomes. M/D: mother/daughter, M: mother, D: daughter. ( G ) Quantification of cells shown in ( E ) and ( F ). Individual data points shown as open circles. REsulting mean and standard deviation show (n=3, N = ≥94). Significant p -values (< 0.05) from a pairwise t-test between G1 and S/G2 populations indicated. ( H ) RPE-1 TRIM37 -/- cells stably expressing FB-TRIM37 were fixed, stained with the indicated antibody, and imaged using 3D-SIM. Two representative images are shown. ( I ) RPE-1 TRIM37 -/- cells stably expressing the indicated construct (FB = FLAG BirA) were pre-extracted, fixed, and stained for the indicated protein. ( J ) Centrosomal TRIM37 signal from cells in ( I ) was quantified. Means from each replicate are shown as open circles. Resulting mean and standard deviation shown (n=3, N≥84). Significant p -values (< 0.05) from Dunnett post hoc test using ‘WT’ as control after one-way ANOVA shown. Note that the results from ( I ) and ( J ) and those in are from the same experiment therefore ‘FLAG-BirA’ and ‘WT’ are duplicated in these panels. See . Figure 3—source data 1. Source data for .

Article Snippet: Antibody , TRIM37 (rabbit, polyclonal) , Bethyl Laboratories , A301-174A , Western blot (1:250) IF (1:250).

Techniques: Stable Transfection, Infection, Expressing, Plasmid Preparation, Staining, Standard Deviation, Western Blot, Construct

( A ) WT RPE-1, TRIM37 -/- (none), and TRIM37 -/- cells expressing FLAG-BirA (FB) or the indicated FB-TRIM37 (WT, C18R, ΔRING) construct were seeded for clonogenic assays and grown in DMSO or the indicated concentration of centrinone B for 14 days. Colony density was quantified and growth compared to that in DMSO determined. Means and standard deviation shown (n=3). Significant p -values (< 0.05) from a Dunnett post hoc test using 'RPE-1' as a control after one-way ANOVA shown. ( B ) WT RPE-1, TRIM37 -/- (pool), and TRIM37 -/- expressing FB or the indicated FB-TRIM37 construct were seeded for clonogenic assays and grown in DMSO or the indicated concentration of centrinone B for 14 days. Colony density was quantified and growth compared to that in DMSO determined. Means from each replicate are shown as open circles. Resulting mean and standard deviation shown (n=3). Significant p -values (< 0.05) from Dunnett post hoc test using ‘WT’ as control after one-way ANOVA shown. Note that the results from this experiment and those in are from the same experiment; therefore ‘WT’, ‘ TRIM37 -/- none’, ‘ TRIM37 -/- FLAG-BirA’, and ‘ TRIM37 -/- WT’ are duplicated in these panels. ( C ) WT or TRIM37 -/- (pools) RPE-1 cells were treated with DMSO (0) or the indicated concentration of centrinone B (nM) for 4 days before fixing and staining for CEP135. CEP135 foci per cell were manually counted. Mean and standard deviation shown (n=3, N≥55 per condition). ( D ) RPE-1 cells were transfected with GFP-PLK4kin + L1 and treated with DMSO or the indicated concentration of centrinone B for 16 hr. The mean and standard deviation among the independent replicates is shown (n=3, N≥12). ( E ) Model showing growth inhibition ‘phases’. Growth is inhibited as a function of centrinone B. Phases dependent on TRIM37 are indicated. Red dots indicate centrosome number. ( F ) RPE-1 TRIM37 -/- cells expressing DOX-inducible TRIM37-3xFLAG or TRIM37 C18R-3xFLAG were seeded for clonogenic assays in the absence and presence of doxycycline and DMSO or the indicated concentration of centrinone B. After incubation for 14 days, colony density was quantified and growth compared to that in DMSO determined. Means from each replicate are shown as open circles. Resulting mean and standard deviation shown (n=4). Significant p -values (< 0.05) from pairwise t-tests comparing -DOX and +DOX samples are shown. ( G ) RPE-1 TRIM37 -/- SASS6 -/- cells expressing DOX-inducible TRIM37-3xFLAG or TRIM37 C18R-3xFLAG were seeded for clonogenic assays in the absence and presence of doxycycline. After incubation for 14 days, colony density was quantified and growth compared to that in DMSO determined. Means from each replicate are shown as open circles. Resulting mean and standard deviation shown (n=3). Significant p -values (< 0.05) from pairwise t-tests comparing -DOX and +DOX samples are shown. ( H ) The indicated RPE-1 line expressing inducible PLK4-3xFLAG were seeded for clonogenic assays in the absence and presence of doxycycline. After incubation for 14 days, colony density was quantified and growth compared to that in DMSO determined. Means from each replicate are shown as open circles. Resulting mean and standard deviation shown (n=3). Significant p -values (<0.05) from pairwise t-tests comparing -DOX and +DOX samples are shown. See . Figure 4—source data 1. Source data for .

Journal: eLife

Article Title: Global cellular response to chemical perturbation of PLK4 activity and abnormal centrosome number

doi: 10.7554/eLife.73944

Figure Lengend Snippet: ( A ) WT RPE-1, TRIM37 -/- (none), and TRIM37 -/- cells expressing FLAG-BirA (FB) or the indicated FB-TRIM37 (WT, C18R, ΔRING) construct were seeded for clonogenic assays and grown in DMSO or the indicated concentration of centrinone B for 14 days. Colony density was quantified and growth compared to that in DMSO determined. Means and standard deviation shown (n=3). Significant p -values (< 0.05) from a Dunnett post hoc test using 'RPE-1' as a control after one-way ANOVA shown. ( B ) WT RPE-1, TRIM37 -/- (pool), and TRIM37 -/- expressing FB or the indicated FB-TRIM37 construct were seeded for clonogenic assays and grown in DMSO or the indicated concentration of centrinone B for 14 days. Colony density was quantified and growth compared to that in DMSO determined. Means from each replicate are shown as open circles. Resulting mean and standard deviation shown (n=3). Significant p -values (< 0.05) from Dunnett post hoc test using ‘WT’ as control after one-way ANOVA shown. Note that the results from this experiment and those in are from the same experiment; therefore ‘WT’, ‘ TRIM37 -/- none’, ‘ TRIM37 -/- FLAG-BirA’, and ‘ TRIM37 -/- WT’ are duplicated in these panels. ( C ) WT or TRIM37 -/- (pools) RPE-1 cells were treated with DMSO (0) or the indicated concentration of centrinone B (nM) for 4 days before fixing and staining for CEP135. CEP135 foci per cell were manually counted. Mean and standard deviation shown (n=3, N≥55 per condition). ( D ) RPE-1 cells were transfected with GFP-PLK4kin + L1 and treated with DMSO or the indicated concentration of centrinone B for 16 hr. The mean and standard deviation among the independent replicates is shown (n=3, N≥12). ( E ) Model showing growth inhibition ‘phases’. Growth is inhibited as a function of centrinone B. Phases dependent on TRIM37 are indicated. Red dots indicate centrosome number. ( F ) RPE-1 TRIM37 -/- cells expressing DOX-inducible TRIM37-3xFLAG or TRIM37 C18R-3xFLAG were seeded for clonogenic assays in the absence and presence of doxycycline and DMSO or the indicated concentration of centrinone B. After incubation for 14 days, colony density was quantified and growth compared to that in DMSO determined. Means from each replicate are shown as open circles. Resulting mean and standard deviation shown (n=4). Significant p -values (< 0.05) from pairwise t-tests comparing -DOX and +DOX samples are shown. ( G ) RPE-1 TRIM37 -/- SASS6 -/- cells expressing DOX-inducible TRIM37-3xFLAG or TRIM37 C18R-3xFLAG were seeded for clonogenic assays in the absence and presence of doxycycline. After incubation for 14 days, colony density was quantified and growth compared to that in DMSO determined. Means from each replicate are shown as open circles. Resulting mean and standard deviation shown (n=3). Significant p -values (< 0.05) from pairwise t-tests comparing -DOX and +DOX samples are shown. ( H ) The indicated RPE-1 line expressing inducible PLK4-3xFLAG were seeded for clonogenic assays in the absence and presence of doxycycline. After incubation for 14 days, colony density was quantified and growth compared to that in DMSO determined. Means from each replicate are shown as open circles. Resulting mean and standard deviation shown (n=3). Significant p -values (<0.05) from pairwise t-tests comparing -DOX and +DOX samples are shown. See . Figure 4—source data 1. Source data for .

Article Snippet: Antibody , TRIM37 (rabbit, polyclonal) , Bethyl Laboratories , A301-174A , Western blot (1:250) IF (1:250).

Techniques: Expressing, Construct, Concentration Assay, Standard Deviation, Staining, Transfection, Inhibition, Incubation

( A ) WT RPE-1 and TRIM37 -/- cells were processed for Western blot and probed for the indicated proteins. Band intensity was quantified and expressed as expression compared to WT cells. Relative intensity from each replicate are shown as open circles. Resulting mean and standard deviation shown (n=3). Data was tested by pairwise t-test between WT and TRIM37 -/- . No significant differences were observed. ( B ) TRIM37 -/- RPE-1 cells stably expressing FLAG-BirA or the indicated FB-TRIM37 protein were processed for Western blot and probed for the indicated proteins. Band intensity was quantified and expressed as expression compared to cells expressing FLAG-BirA. Relative intensity from each replicate are shown as open circles. Resulting mean and standard deviation shown (n=3). Significant p -values (< 0.05) from Dunnett post hoc test using FLAG-BirA as a control after a one-way ANOVA are shown. The band intensity from FLAG-BirA cells was set to ‘1’ and is omitted from the plot for clarity. ( C ) WT RPE-1 cells expressing doxycline-inducible TRIM37-3xFLAG (WT) or TRIM37 C18R-3xFLAG (C18R) were induced with doxycycline for 0, 4, or 8 hr. At each time point, extracts were prepared and analyzed by Western blot for the indicated protein (right). Ponc.S indicates equal loading. CEP192 abundance was quantified and normalized to the intensity at time 0 hr (bottom). Mean and standard deviation shown (n=3). Significant p -values (< 0.05) from Dunnett post hoc test using time 0 hr as a control after a one-way ANOVA are shown. ( D ) Cells from ( C ) were also fixed and immunostained for the indicated proteins. The centrosomal intensity from mitotic cells was determined. Intensity values were normalized to 0 hr. Means from each replicate are shown as open circles. Resulting mean and standard deviation shown (n=3, N=60). Significant p -values (< 0.05) from Dunnett post hoc test using time 0 hr as a control after a one-way ANOVA are shown. ( E ) RPE-1 TRIM37 -/- cells were fixed and stained for CEP120 and the indicated proteins. ( F ) RPE-1 (WT) or TRIM37 -/- cells stably expressing FLAG-BirA or the indicated FB-TRIM37 protein were fixed and stained for the indicated protein. Arrowhead indicates centrosome defined by CEP192. Caret mark indicates ectopic structure defined by CEP120. See . Figure 6—source data 1. Source data for .

Journal: eLife

Article Title: Global cellular response to chemical perturbation of PLK4 activity and abnormal centrosome number

doi: 10.7554/eLife.73944

Figure Lengend Snippet: ( A ) WT RPE-1 and TRIM37 -/- cells were processed for Western blot and probed for the indicated proteins. Band intensity was quantified and expressed as expression compared to WT cells. Relative intensity from each replicate are shown as open circles. Resulting mean and standard deviation shown (n=3). Data was tested by pairwise t-test between WT and TRIM37 -/- . No significant differences were observed. ( B ) TRIM37 -/- RPE-1 cells stably expressing FLAG-BirA or the indicated FB-TRIM37 protein were processed for Western blot and probed for the indicated proteins. Band intensity was quantified and expressed as expression compared to cells expressing FLAG-BirA. Relative intensity from each replicate are shown as open circles. Resulting mean and standard deviation shown (n=3). Significant p -values (< 0.05) from Dunnett post hoc test using FLAG-BirA as a control after a one-way ANOVA are shown. The band intensity from FLAG-BirA cells was set to ‘1’ and is omitted from the plot for clarity. ( C ) WT RPE-1 cells expressing doxycline-inducible TRIM37-3xFLAG (WT) or TRIM37 C18R-3xFLAG (C18R) were induced with doxycycline for 0, 4, or 8 hr. At each time point, extracts were prepared and analyzed by Western blot for the indicated protein (right). Ponc.S indicates equal loading. CEP192 abundance was quantified and normalized to the intensity at time 0 hr (bottom). Mean and standard deviation shown (n=3). Significant p -values (< 0.05) from Dunnett post hoc test using time 0 hr as a control after a one-way ANOVA are shown. ( D ) Cells from ( C ) were also fixed and immunostained for the indicated proteins. The centrosomal intensity from mitotic cells was determined. Intensity values were normalized to 0 hr. Means from each replicate are shown as open circles. Resulting mean and standard deviation shown (n=3, N=60). Significant p -values (< 0.05) from Dunnett post hoc test using time 0 hr as a control after a one-way ANOVA are shown. ( E ) RPE-1 TRIM37 -/- cells were fixed and stained for CEP120 and the indicated proteins. ( F ) RPE-1 (WT) or TRIM37 -/- cells stably expressing FLAG-BirA or the indicated FB-TRIM37 protein were fixed and stained for the indicated protein. Arrowhead indicates centrosome defined by CEP192. Caret mark indicates ectopic structure defined by CEP120. See . Figure 6—source data 1. Source data for .

Article Snippet: Antibody , TRIM37 (rabbit, polyclonal) , Bethyl Laboratories , A301-174A , Western blot (1:250) IF (1:250).

Techniques: Western Blot, Expressing, Standard Deviation, Stable Transfection, Staining

( A ) WT RPE-1 and TRIM37 -/- cells were processed for Western blot and probed for the indicated protein. Ponc.S indicates total protein. Quantification in . ( B ) TRIM37 -/- RPE-1 cells stably expressing FLAG-BirA (FB) or the indicated FB-TRIM37 protein were processed for Western blot and probed for the indicated protein. Ponc.S indicates total protein. Quantification in . ( C ) RPE-1 TRIM37 -/- cells expressing inducible TRIM37 or TRIM37 C18R were induced with doxycycline for the indicated time. Cells were fixed and stained for the indicated protein. Sample images of mitotic cells quantified in .

Journal: eLife

Article Title: Global cellular response to chemical perturbation of PLK4 activity and abnormal centrosome number

doi: 10.7554/eLife.73944

Figure Lengend Snippet: ( A ) WT RPE-1 and TRIM37 -/- cells were processed for Western blot and probed for the indicated protein. Ponc.S indicates total protein. Quantification in . ( B ) TRIM37 -/- RPE-1 cells stably expressing FLAG-BirA (FB) or the indicated FB-TRIM37 protein were processed for Western blot and probed for the indicated protein. Ponc.S indicates total protein. Quantification in . ( C ) RPE-1 TRIM37 -/- cells expressing inducible TRIM37 or TRIM37 C18R were induced with doxycycline for the indicated time. Cells were fixed and stained for the indicated protein. Sample images of mitotic cells quantified in .

Article Snippet: Antibody , TRIM37 (rabbit, polyclonal) , Bethyl Laboratories , A301-174A , Western blot (1:250) IF (1:250).

Techniques: Western Blot, Stable Transfection, Expressing, Staining

( A ) TRIM37 domain schematic. Constructs used for structure-function experiments indicated below. ( B ) RPE-1cells were transfected to express Myc-PLK4 and FLAG-BirA or the indicated FB-TRIM37 fusion protein (top). Cells were lysed and subjected to anti-FLAG immunoprecipitation. Input and immunoprecipitates were analyzed by immunoblotting for the FLAG-BirA fusions (FLAG) or for Myc-PLK4. Ponc.S indicates total protein. * indicates position of FLAG-Cas9. ( C ) WT RPE-1, TRIM37 -/- and TRIM37 -/- expressing FB or the indicated FB-TRIM37 construct were seeded for clonogenic assays and grown in DMSO or the indicated concentration of centrinone B for 14 days. Colony density was quantified and growth compared to that in DMSO determined. Means from each replicate are shown as open circles. Resulting mean and standard deviation shown (n=3). Significant p -values (< 0.05) from Dunnett post hoc test using ‘WT’ as a control after one-way ANOVA shown. Note that the results from this experiment and those in are from the same experiment; therefore ‘WT’, ‘ TRIM37 -/- none’, ‘ TRIM37 -/- FLAG-BirA’, and ‘ TRIM37 -/- WT’ are duplicated in these panels. ( D ) HEK293T cells transfected to express Myc-PLK4 and the indicated protein (top) were grown overnight and subsequently treated with DMSO or MLN4924 for 22 hr and MG132 for the final 4 hr. Cell extracts were prepared and probed by Western blot using the indicated antibodies. Ponc.S indicates total protein. ( E ) HEK293T cells were transfected to express Myc-PLK4, HA-Ub, and the indicated protein (top). Cells were harvested after 48 hr and subjected to immunoprecipitation using anti-Myc antibodies. Input and immunoprecipitates were analyzed by immunoblotting for PLK4 and HA-Ub. Ponc.S indicates total protein. ( F ) HEK293T cells were transfected to express Myc-PLK4 and eGFP or the indicated T7-TRIM37 protein (top) for 48 hr. MG132 was added for the final 4 hr. Lysates were mock treated (-λ) or incubated with λ-phosphatase (+λ) and subsequently subjected to immunoblot for PLK4. ( CB ) indicates total protein. ( G ) HEK293T cells were transfected to express Myc-PLK4 and T7-TRIM37 Δ505–709 for 48 hr. Cells were treated with the indicated inhibitor (top) for 3 or 6 hr and analyzed by immunoblot for PLK4. Ponc.S indicates total protein. See . Figure 7—source data 1. Source data for .

Journal: eLife

Article Title: Global cellular response to chemical perturbation of PLK4 activity and abnormal centrosome number

doi: 10.7554/eLife.73944

Figure Lengend Snippet: ( A ) TRIM37 domain schematic. Constructs used for structure-function experiments indicated below. ( B ) RPE-1cells were transfected to express Myc-PLK4 and FLAG-BirA or the indicated FB-TRIM37 fusion protein (top). Cells were lysed and subjected to anti-FLAG immunoprecipitation. Input and immunoprecipitates were analyzed by immunoblotting for the FLAG-BirA fusions (FLAG) or for Myc-PLK4. Ponc.S indicates total protein. * indicates position of FLAG-Cas9. ( C ) WT RPE-1, TRIM37 -/- and TRIM37 -/- expressing FB or the indicated FB-TRIM37 construct were seeded for clonogenic assays and grown in DMSO or the indicated concentration of centrinone B for 14 days. Colony density was quantified and growth compared to that in DMSO determined. Means from each replicate are shown as open circles. Resulting mean and standard deviation shown (n=3). Significant p -values (< 0.05) from Dunnett post hoc test using ‘WT’ as a control after one-way ANOVA shown. Note that the results from this experiment and those in are from the same experiment; therefore ‘WT’, ‘ TRIM37 -/- none’, ‘ TRIM37 -/- FLAG-BirA’, and ‘ TRIM37 -/- WT’ are duplicated in these panels. ( D ) HEK293T cells transfected to express Myc-PLK4 and the indicated protein (top) were grown overnight and subsequently treated with DMSO or MLN4924 for 22 hr and MG132 for the final 4 hr. Cell extracts were prepared and probed by Western blot using the indicated antibodies. Ponc.S indicates total protein. ( E ) HEK293T cells were transfected to express Myc-PLK4, HA-Ub, and the indicated protein (top). Cells were harvested after 48 hr and subjected to immunoprecipitation using anti-Myc antibodies. Input and immunoprecipitates were analyzed by immunoblotting for PLK4 and HA-Ub. Ponc.S indicates total protein. ( F ) HEK293T cells were transfected to express Myc-PLK4 and eGFP or the indicated T7-TRIM37 protein (top) for 48 hr. MG132 was added for the final 4 hr. Lysates were mock treated (-λ) or incubated with λ-phosphatase (+λ) and subsequently subjected to immunoblot for PLK4. ( CB ) indicates total protein. ( G ) HEK293T cells were transfected to express Myc-PLK4 and T7-TRIM37 Δ505–709 for 48 hr. Cells were treated with the indicated inhibitor (top) for 3 or 6 hr and analyzed by immunoblot for PLK4. Ponc.S indicates total protein. See . Figure 7—source data 1. Source data for .

Article Snippet: Antibody , TRIM37 (rabbit, polyclonal) , Bethyl Laboratories , A301-174A , Western blot (1:250) IF (1:250).

Techniques: Construct, Transfection, Immunoprecipitation, Western Blot, Expressing, Concentration Assay, Standard Deviation, Incubation

PLK4 activity decreases in a dose-dependent manner upon centrinone B addition. TRIM37 promotes PLK4 auto-phosphorylation (orange circles) outside the phosphodegron region (purple circles). PLK4 inhibition initially results in TRIM37-independent growth arrest. Continued addition of centrinone B results in centrosome overduplication that is detected by the ANKRD26/PIDDosome pathway in addition to a TRIM37-dependent growth arrest pathway. Complete inhibition of PLK4 results in TRIM37-dependent growth arrest. TRIM37 also prevents the appearance of CNTROB-dependent aggregates. We hypothesize that these aggregates might affect p53/p21 activation (dotted lines) (created with https://biorender.com/ ).

Journal: eLife

Article Title: Global cellular response to chemical perturbation of PLK4 activity and abnormal centrosome number

doi: 10.7554/eLife.73944

Figure Lengend Snippet: PLK4 activity decreases in a dose-dependent manner upon centrinone B addition. TRIM37 promotes PLK4 auto-phosphorylation (orange circles) outside the phosphodegron region (purple circles). PLK4 inhibition initially results in TRIM37-independent growth arrest. Continued addition of centrinone B results in centrosome overduplication that is detected by the ANKRD26/PIDDosome pathway in addition to a TRIM37-dependent growth arrest pathway. Complete inhibition of PLK4 results in TRIM37-dependent growth arrest. TRIM37 also prevents the appearance of CNTROB-dependent aggregates. We hypothesize that these aggregates might affect p53/p21 activation (dotted lines) (created with https://biorender.com/ ).

Article Snippet: Antibody , TRIM37 (rabbit, polyclonal) , Bethyl Laboratories , A301-174A , Western blot (1:250) IF (1:250).

Techniques: Activity Assay, Inhibition, Activation Assay

Journal: eLife

Article Title: Global cellular response to chemical perturbation of PLK4 activity and abnormal centrosome number

doi: 10.7554/eLife.73944

Figure Lengend Snippet:

Article Snippet: Antibody , TRIM37 (rabbit, polyclonal) , Bethyl Laboratories , A301-174A , Western blot (1:250) IF (1:250).

Techniques: Clone Assay, Western Blot, Recombinant, Plasmid Preparation, Sequencing, Luciferase, Immunoprecipitation, Software, Transfection, Mutagenesis

( A, B ) Western blot of lysates from HeLa cells ( A ) or HeLa Centrin-1:GFP cells ( B ) transfected with siRNAs against TRIM37 and probed with antibodies against TRIM37 (top) or α-tubulin as loading control (bottom). Select molecular weight markers are indicated in kDa in this and other western blot panels. ( C ) HeLa cells expressing Centrin-1:GFP immunostained for GFP, HsSAS-6 and CEP63. Nuclear contours are drawn with dashed yellow lines. In this and other supplement figure panels, scale bars correspond to 5 μm, unless indicated otherwise. ( D ) HeLa cells immunostained for Centrin-2 and CP110. Left: G1 cell, with two resident centrioles, right: S/G2 cell with two centriole pairs, each with one resident centriole and one procentriole. ( E ) Western blot of cell lysates from control and Mulibrey patient (P-1, P-2) fibroblasts probed with antibodies against TRIM37 (top) or α-tubulin as loading control (bottom). The arrow indicates TRIM37, the asterisk a non-specific band.

Journal: eLife

Article Title: TRIM37 prevents formation of centriolar protein assemblies by regulating Centrobin

doi: 10.7554/eLife.62640

Figure Lengend Snippet: ( A, B ) Western blot of lysates from HeLa cells ( A ) or HeLa Centrin-1:GFP cells ( B ) transfected with siRNAs against TRIM37 and probed with antibodies against TRIM37 (top) or α-tubulin as loading control (bottom). Select molecular weight markers are indicated in kDa in this and other western blot panels. ( C ) HeLa cells expressing Centrin-1:GFP immunostained for GFP, HsSAS-6 and CEP63. Nuclear contours are drawn with dashed yellow lines. In this and other supplement figure panels, scale bars correspond to 5 μm, unless indicated otherwise. ( D ) HeLa cells immunostained for Centrin-2 and CP110. Left: G1 cell, with two resident centrioles, right: S/G2 cell with two centriole pairs, each with one resident centriole and one procentriole. ( E ) Western blot of cell lysates from control and Mulibrey patient (P-1, P-2) fibroblasts probed with antibodies against TRIM37 (top) or α-tubulin as loading control (bottom). The arrow indicates TRIM37, the asterisk a non-specific band.

Article Snippet: Primary antibodies were 1:1000 rabbit anti-TRIM37 (A301-174A; Bethyl Laboratories), 1:30,000 mouse anti-α-tubulin (T6199; Sigma-Aldrich), 1:500 rabbit anti-Centrobin (HPA023321; Atlas), and 1:20,000 mouse anti-HSP70 (sc-24; Santa Cruz).

Techniques: Western Blot, Transfection, Molecular Weight, Expressing

( A ) Relevant images from wide-field time-lapse recordings of HeLa cells expressing Centrin-1:GFP and depleted of TRIM37 for 48 hr before imaging onset (10 min time frame). Yellow arrows point to two foci appearing close to resident centrioles (8/13 extra foci in 11 cells), orange arrow to one focus appearing away from resident centrioles (5/13 extra foci). Solid arrows indicate first occurrence of foci, dashed arrows their continued presence. Time is indicated in h:min since imaging onset. Note that the intensity of extra Centrin-1:GFP foci was typically weaker than that of regular centrioles, especially in the early assembly stages. Note also resident centriole and procentriole appearing in the field of view at the bottom right in Cell 1, 9:20. In this and other Figure panels, scale bars correspond to 5 μm, unless indicated otherwise. ( B ) HeLa cells expressing Centrin-1:GFP upon treatment with TRIM37 siRNAs or upon RO3306 addition for 48 hr. Cells were immunostained for GFP, HsSAS-6 and CEP63. Nuclear contours are drawn with dashed yellow lines. In this and subsequent figures, magnified images from indicated numbered regions are shown. ( C ) Corresponding percentage of cells with extra Centrin-1:GFP foci that also harbor CEP63 and/or HsSAS-6. Note that extra Centrin-1:GFP foci could be positive for both Cep63 and HsSAS-6 in RO3306-treated cells. Chart shows the average and SDs from two independent experiments (n = 50 cells each). Here and in other charts of this figure, two conditions that do not share the same letter are significantly different from each other, with p<0.05; unpaired Student’s t-test; see for exact p values. ( D ) HeLa cells depleted of TRIM37 and immunostained for Centrin-2 plus CP110, illustrating a case with an extra single focus (left, inset 1) and one with an extra pair of foci (right). DNA is shown in blue in this and all other figure panels unless stated otherwise. ( E ) Corresponding percentage of interphase cells with extra single focus or extra pairs of foci at indicated times after TRIM37 siRNA transfection. Chart shows the average and SDs from three independent experiments (n = 50 cells each). ( F ) Microtubule depolymerization-regrowth experiment in mitotic HeLa cells treated with control or TRIM37 siRNAs. Microtubules were depolymerized by a 30-min cold shock followed by 1–2 min at room temperature before fixation and immunostaining for Centrin-2 and α-tubulin. ( G ) Corresponding percentage of mitotic cells with >2 MTOCs. Chart shows the average and SDs from three independent experiments (n = 50 cells each). Note that ~40% of the extra Centrin-2 foci observed in mitosis did not nucleate microtubules, as illustrated for two of them in inset 1 (siTRIM37); data from n = 40 Cenpas in each of the three independent experiments. Source data for panels C, E, and G can be found in . Figure 1—source data 1. Source data for figure panels: , .

Journal: eLife

Article Title: TRIM37 prevents formation of centriolar protein assemblies by regulating Centrobin

doi: 10.7554/eLife.62640

Figure Lengend Snippet: ( A ) Relevant images from wide-field time-lapse recordings of HeLa cells expressing Centrin-1:GFP and depleted of TRIM37 for 48 hr before imaging onset (10 min time frame). Yellow arrows point to two foci appearing close to resident centrioles (8/13 extra foci in 11 cells), orange arrow to one focus appearing away from resident centrioles (5/13 extra foci). Solid arrows indicate first occurrence of foci, dashed arrows their continued presence. Time is indicated in h:min since imaging onset. Note that the intensity of extra Centrin-1:GFP foci was typically weaker than that of regular centrioles, especially in the early assembly stages. Note also resident centriole and procentriole appearing in the field of view at the bottom right in Cell 1, 9:20. In this and other Figure panels, scale bars correspond to 5 μm, unless indicated otherwise. ( B ) HeLa cells expressing Centrin-1:GFP upon treatment with TRIM37 siRNAs or upon RO3306 addition for 48 hr. Cells were immunostained for GFP, HsSAS-6 and CEP63. Nuclear contours are drawn with dashed yellow lines. In this and subsequent figures, magnified images from indicated numbered regions are shown. ( C ) Corresponding percentage of cells with extra Centrin-1:GFP foci that also harbor CEP63 and/or HsSAS-6. Note that extra Centrin-1:GFP foci could be positive for both Cep63 and HsSAS-6 in RO3306-treated cells. Chart shows the average and SDs from two independent experiments (n = 50 cells each). Here and in other charts of this figure, two conditions that do not share the same letter are significantly different from each other, with p<0.05; unpaired Student’s t-test; see for exact p values. ( D ) HeLa cells depleted of TRIM37 and immunostained for Centrin-2 plus CP110, illustrating a case with an extra single focus (left, inset 1) and one with an extra pair of foci (right). DNA is shown in blue in this and all other figure panels unless stated otherwise. ( E ) Corresponding percentage of interphase cells with extra single focus or extra pairs of foci at indicated times after TRIM37 siRNA transfection. Chart shows the average and SDs from three independent experiments (n = 50 cells each). ( F ) Microtubule depolymerization-regrowth experiment in mitotic HeLa cells treated with control or TRIM37 siRNAs. Microtubules were depolymerized by a 30-min cold shock followed by 1–2 min at room temperature before fixation and immunostaining for Centrin-2 and α-tubulin. ( G ) Corresponding percentage of mitotic cells with >2 MTOCs. Chart shows the average and SDs from three independent experiments (n = 50 cells each). Note that ~40% of the extra Centrin-2 foci observed in mitosis did not nucleate microtubules, as illustrated for two of them in inset 1 (siTRIM37); data from n = 40 Cenpas in each of the three independent experiments. Source data for panels C, E, and G can be found in . Figure 1—source data 1. Source data for figure panels: , .

Techniques: Expressing, Imaging, Transfection, Immunostaining

( A ) HeLa cells expressing TRIM37:GFP or TRIM37 tagged with a nuclear export signal and GFP (TRIM37:NES:GFP) immunostained for GFP. An inset of the indicated area with modified brightness and contrast is shown at the bottom of each image (1*). ( B ) Quantification of extra number of CP110 foci in HeLa cells treated with control or TRIM37 siRNAs and transfected with indicated plasmids (pcDNA3: parental vector). Cells were immunostained for GFP and CP110. Chart shows the average and SDs from two independent experiments (n = 50 cells each). Two conditions that do not share the same letter are significantly different from each other, with p<0.05; unpaired Student’s t-test; see for exact p values. ( C ) HeLa (left) and U2OS (right) cells transfected with TRIM37:GFP, and immunostained for GFP plus γ-tubulin. ( D ) HeLa cells transfected with TRIM37:GFP, and immunostained for GFP plus Centrobin. Cells with a single Centrobin focus (left) were classified as being in G1, cells with two or three Centrobin foci (right) as being in S/G2. ( E ) Corresponding percentage of cells in G1 or S/G2 exhibiting TRIM37:GFP at centrosomes. n = 50 cells, single experiment. ( F, G ) High magnification confocal images of TRIM37:GFP localization with respect to indicated centriolar markers; HeLa cells were fixed 24 hr after transfection in this case. Scale bar 500 nm. ( H ) Microtubule depolymerization-regrowth experiment of mitotic HeLa cells treated with control or TRIM37 siRNAs. Microtubules were depolymerized by a 30-min cold shock, fixed and immunostained for Centrin-2 and α-tubulin. Source data for panels B and E can be found in .

Journal: eLife

Article Title: TRIM37 prevents formation of centriolar protein assemblies by regulating Centrobin

doi: 10.7554/eLife.62640

Figure Lengend Snippet: ( A ) HeLa cells expressing TRIM37:GFP or TRIM37 tagged with a nuclear export signal and GFP (TRIM37:NES:GFP) immunostained for GFP. An inset of the indicated area with modified brightness and contrast is shown at the bottom of each image (1*). ( B ) Quantification of extra number of CP110 foci in HeLa cells treated with control or TRIM37 siRNAs and transfected with indicated plasmids (pcDNA3: parental vector). Cells were immunostained for GFP and CP110. Chart shows the average and SDs from two independent experiments (n = 50 cells each). Two conditions that do not share the same letter are significantly different from each other, with p<0.05; unpaired Student’s t-test; see for exact p values. ( C ) HeLa (left) and U2OS (right) cells transfected with TRIM37:GFP, and immunostained for GFP plus γ-tubulin. ( D ) HeLa cells transfected with TRIM37:GFP, and immunostained for GFP plus Centrobin. Cells with a single Centrobin focus (left) were classified as being in G1, cells with two or three Centrobin foci (right) as being in S/G2. ( E ) Corresponding percentage of cells in G1 or S/G2 exhibiting TRIM37:GFP at centrosomes. n = 50 cells, single experiment. ( F, G ) High magnification confocal images of TRIM37:GFP localization with respect to indicated centriolar markers; HeLa cells were fixed 24 hr after transfection in this case. Scale bar 500 nm. ( H ) Microtubule depolymerization-regrowth experiment of mitotic HeLa cells treated with control or TRIM37 siRNAs. Microtubules were depolymerized by a 30-min cold shock, fixed and immunostained for Centrin-2 and α-tubulin. Source data for panels B and E can be found in .

Techniques: Expressing, Modification, Transfection, Plasmid Preparation

( A–E ) Ultrastructure expansion microscopy (U-ExM) confocal images of control ( A ) or TRIM37 ( B–E ) depleted RPE-1 cells expressing Centrin-1:GFP, and immunostained for GFP, CEP152 as well as acetylated tubulin. Yellow arrows point to Cenpas lacking CEP152 and acetylated tubulin, white arrows to those harboring both proteins, but with an unusual distribution. Scale bar 500 nm. ( F–J ) CLEM analysis of HeLa cell (cell three in ) expressing Centrin-1:GFP and depleted of TRIM37. Maximal intensity projection of wide-field microcopy image covering the entire cell volume ( F ), and magnified insets from the light microscopy images above the corresponding 50 nm section EM images ( G–J ), with white arrows pointing to relevant Centrin-1:GFP focus. Scale bars: 5 μm in F, 500 nm in G. Here and in panels K-M, green and pink dashed lines surround centriole-related and tiger structures, respectively. Filled orange lines surround resident centrioles, which could be recognized when going through all the sections encompassing the organelle. ( K–M ) Centriole-related (K, cell seven in ; L, cell seven in ), and tiger structure (M, cell two in ). Scale bar is 500 nm.

Journal: eLife

Article Title: TRIM37 prevents formation of centriolar protein assemblies by regulating Centrobin

doi: 10.7554/eLife.62640

Figure Lengend Snippet: ( A–E ) Ultrastructure expansion microscopy (U-ExM) confocal images of control ( A ) or TRIM37 ( B–E ) depleted RPE-1 cells expressing Centrin-1:GFP, and immunostained for GFP, CEP152 as well as acetylated tubulin. Yellow arrows point to Cenpas lacking CEP152 and acetylated tubulin, white arrows to those harboring both proteins, but with an unusual distribution. Scale bar 500 nm. ( F–J ) CLEM analysis of HeLa cell (cell three in ) expressing Centrin-1:GFP and depleted of TRIM37. Maximal intensity projection of wide-field microcopy image covering the entire cell volume ( F ), and magnified insets from the light microscopy images above the corresponding 50 nm section EM images ( G–J ), with white arrows pointing to relevant Centrin-1:GFP focus. Scale bars: 5 μm in F, 500 nm in G. Here and in panels K-M, green and pink dashed lines surround centriole-related and tiger structures, respectively. Filled orange lines surround resident centrioles, which could be recognized when going through all the sections encompassing the organelle. ( K–M ) Centriole-related (K, cell seven in ; L, cell seven in ), and tiger structure (M, cell two in ). Scale bar is 500 nm.

Techniques: Microscopy, Expressing, Light Microscopy

( A ) Centriolar and centrosomal proteins analyzed by immunofluorescence upon TRIM37 depletion. See Materials and methods for antibodies utilized. ( B ) Quantification of frequency of Centrobin assemblies in HeLa cells depleted of TRIM37. Chart shows the average and SDs from three independent experiments (n = 50 cells each). Here and in other charts of this figure, two conditions that do not share the same letter are significantly different from each other, with p<0.05; unpaired Student’s t-test; see for exact p values. ( C–E ) HeLa cells depleted of TRIM37 and immunostained with antibodies against Centrobin and Rootletin ( C ), Centrobin and CEP68 ( D ), Centrobin and HsSAS-6 ( E ). ( F ) HeLa cells treated with control, TRIM37 or simultaneously with both TRIM37 and SPICE siRNAs, immunostained for SPICE and Centrobin. ( G ) HeLa cell expressing SPICE:GFP immunostained for GFP and Centrobin. ( H ) RPE-1 and RPE-1 TRIM37 knock-out cells immunostained with antibodies against Centrobin and PLK4 . ( I ) Quantification of mean PLK4 intensity in Centrobin structures in HeLa cells depleted of TRIM37 or double depleted of TRIM37 and PLK4 as in . Chart shows the average and SDs from two independent experiments. Approximately 50 Centrobin structures were quantified per experiment and condition. ( J ) Quantification of area of Centrobin assemblies in HeLa cells depleted of TRIM37 or simultaneously of both TRIM37 and PLK4, as in . Chart shows the average and SDs from two independent experiments. Approximately 50 Centrobin structures were quantified per experiment and condition. Source data for panels B, I, and J can be found in .

Journal: eLife

Article Title: TRIM37 prevents formation of centriolar protein assemblies by regulating Centrobin

doi: 10.7554/eLife.62640

Figure Lengend Snippet: ( A ) Centriolar and centrosomal proteins analyzed by immunofluorescence upon TRIM37 depletion. See Materials and methods for antibodies utilized. ( B ) Quantification of frequency of Centrobin assemblies in HeLa cells depleted of TRIM37. Chart shows the average and SDs from three independent experiments (n = 50 cells each). Here and in other charts of this figure, two conditions that do not share the same letter are significantly different from each other, with p<0.05; unpaired Student’s t-test; see for exact p values. ( C–E ) HeLa cells depleted of TRIM37 and immunostained with antibodies against Centrobin and Rootletin ( C ), Centrobin and CEP68 ( D ), Centrobin and HsSAS-6 ( E ). ( F ) HeLa cells treated with control, TRIM37 or simultaneously with both TRIM37 and SPICE siRNAs, immunostained for SPICE and Centrobin. ( G ) HeLa cell expressing SPICE:GFP immunostained for GFP and Centrobin. ( H ) RPE-1 and RPE-1 TRIM37 knock-out cells immunostained with antibodies against Centrobin and PLK4 . ( I ) Quantification of mean PLK4 intensity in Centrobin structures in HeLa cells depleted of TRIM37 or double depleted of TRIM37 and PLK4 as in . Chart shows the average and SDs from two independent experiments. Approximately 50 Centrobin structures were quantified per experiment and condition. ( J ) Quantification of area of Centrobin assemblies in HeLa cells depleted of TRIM37 or simultaneously of both TRIM37 and PLK4, as in . Chart shows the average and SDs from two independent experiments. Approximately 50 Centrobin structures were quantified per experiment and condition. Source data for panels B, I, and J can be found in .

Techniques: Immunofluorescence, Expressing, Knock-Out

$( A ) HeLa cells in G2 or mitosis (M), as indicated in the upper left corners of the images, treated with control or TRIM37 siRNAs, and immunostained for CP110 plus Centrobin. ( B ) High-magnification confocal view of cells treated with control or TRIM37 siRNAs immunostained for Centrobin and CP110. Arrow points to elongated Centrobin assembly. Scale bar 1 μm. ( C ) Number of Centrobin structures in HeLa cells depleted of TRIM37. Chart shows the average and SDs from three independent experiments (n = 50 cells each). ( D ) HeLa cells were first transfected with PLK4 or Centrobin siRNAs and 24 hr thereafter transfected again with TRIM37 siRNAs. Cells were fixed 72 hr after first transfection and stained with Centrobin and PLK4 antibodies . ( E ) Corresponding percentage of cells bearing Centrobin structures. Chart shows the average and SDs from three independent experiments (n = 50 cells each). Here and in other charts of this figure, two conditions that do not share the same letter are significantly different from each other, with p<0.05; unpaired Student’s t-test; see for exact p values. ( F ) U-ExM coupled to STED super-resolution microscopy of RPE-1 cells immmunostained for CEP152, Centrin-1 and Centrobin. White arrows point to Cenpas in close proximity to Centrobin assembly. Scale bars are 250 nm. ( G ) Box-and-whisker plot of inter stripe distances in TEM tiger structures (n = 53 from five tiger structures) and U-ExM Centrobin structures (n = 30 from three Centrobin structures). U-ExM-induced sample expansion was taken into consideration to compare TEM vs. U-ExM inter stripe measurements. Box plots show median, interquartile range (10–90 percentile) and SDs. ( H ) Western blot of lysates from HeLa cells treated with control or TRIM37 siRNAs probed with antibodies against Centrobin (top) or HSP70 as loading control (bottom). ( I ) Western blot of soluble (S) or insoluble (P, for pellet) fractions of lysates from HeLa cells treated with control or TRIM37 siRNAs, probed with antibodies against Centrobin (top) or α-tubulin as loading control (bottom). Note that Centrobin in the insoluble fraction migrates slower upon TRIM37 depletion, suggestive of some posttranslational modification. ( J ) Western blot of total Centrobin protein levels in control and TRIM37-depleted HeLa cells treated with cycloheximide (CHX) for indicated time in hours (h), probed with antibodies against Centrobin (top) or α-tubulin as loading control (bottom). Note that the amount of lysate loaded for the TRIM37-depleted sample was ~50% of that loaded for the siControl condition in this case. ( K ) Quantification of relative Centrobin protein levels from western blots such as the one shown in J. Chart shows the average and SDs from two independent experiments. Lower-case and upper-case letters above the charts reflect comparisons of two distinct data sets. Source data for panels C, E, G, and K can be found in . Figure 4—source data 1. Source data for figure panels: , and .$

Journal: eLife

Article Title: TRIM37 prevents formation of centriolar protein assemblies by regulating Centrobin

doi: 10.7554/eLife.62640

Figure Lengend Snippet: ( A ) HeLa cells in G2 or mitosis (M), as indicated in the upper left corners of the images, treated with control or TRIM37 siRNAs, and immunostained for CP110 plus Centrobin. ( B ) High-magnification confocal view of cells treated with control or TRIM37 siRNAs immunostained for Centrobin and CP110. Arrow points to elongated Centrobin assembly. Scale bar 1 μm. ( C ) Number of Centrobin structures in HeLa cells depleted of TRIM37. Chart shows the average and SDs from three independent experiments (n = 50 cells each). ( D ) HeLa cells were first transfected with PLK4 or Centrobin siRNAs and 24 hr thereafter transfected again with TRIM37 siRNAs. Cells were fixed 72 hr after first transfection and stained with Centrobin and PLK4 antibodies . ( E ) Corresponding percentage of cells bearing Centrobin structures. Chart shows the average and SDs from three independent experiments (n = 50 cells each). Here and in other charts of this figure, two conditions that do not share the same letter are significantly different from each other, with p<0.05; unpaired Student’s t-test; see for exact p values. ( F ) U-ExM coupled to STED super-resolution microscopy of RPE-1 cells immmunostained for CEP152, Centrin-1 and Centrobin. White arrows point to Cenpas in close proximity to Centrobin assembly. Scale bars are 250 nm. ( G ) Box-and-whisker plot of inter stripe distances in TEM tiger structures (n = 53 from five tiger structures) and U-ExM Centrobin structures (n = 30 from three Centrobin structures). U-ExM-induced sample expansion was taken into consideration to compare TEM vs. U-ExM inter stripe measurements. Box plots show median, interquartile range (10–90 percentile) and SDs. ( H ) Western blot of lysates from HeLa cells treated with control or TRIM37 siRNAs probed with antibodies against Centrobin (top) or HSP70 as loading control (bottom). ( I ) Western blot of soluble (S) or insoluble (P, for pellet) fractions of lysates from HeLa cells treated with control or TRIM37 siRNAs, probed with antibodies against Centrobin (top) or α-tubulin as loading control (bottom). Note that Centrobin in the insoluble fraction migrates slower upon TRIM37 depletion, suggestive of some posttranslational modification. ( J ) Western blot of total Centrobin protein levels in control and TRIM37-depleted HeLa cells treated with cycloheximide (CHX) for indicated time in hours (h), probed with antibodies against Centrobin (top) or α-tubulin as loading control (bottom). Note that the amount of lysate loaded for the TRIM37-depleted sample was ~50% of that loaded for the siControl condition in this case. ( K ) Quantification of relative Centrobin protein levels from western blots such as the one shown in J. Chart shows the average and SDs from two independent experiments. Lower-case and upper-case letters above the charts reflect comparisons of two distinct data sets. Source data for panels C, E, G, and K can be found in . Figure 4—source data 1. Source data for figure panels: , and .

Techniques: Transfection, Staining, Microscopy, Whisker Assay, Western Blot, Modification

( A ) Quantitative real time PCR of TRIM37 and Centrobin mRNA in HeLa cells treated with control or TRIM37 siRNAs. Average of three independent experiments. In the statistical analysis, lower-case and upper-case letters indicate two independent set of data analyzed. Two conditions that do not share the same letter are significantly different from each other, with p<0.05; unpaired Student’s t-test; see for exact p values. ( B ) HeLa cell (left) and HeLa cell overexpressing Centrobin:GFP (right), immunostained for GFP and CP110. ( C ) Confocal images of HeLa cells overexpressing TRIM37:GFP immunostained with antibodies against GFP and Centrobin. Source data for panel A can be found in .

Journal: eLife

Article Title: TRIM37 prevents formation of centriolar protein assemblies by regulating Centrobin

doi: 10.7554/eLife.62640

Figure Lengend Snippet: ( A ) Quantitative real time PCR of TRIM37 and Centrobin mRNA in HeLa cells treated with control or TRIM37 siRNAs. Average of three independent experiments. In the statistical analysis, lower-case and upper-case letters indicate two independent set of data analyzed. Two conditions that do not share the same letter are significantly different from each other, with p<0.05; unpaired Student’s t-test; see for exact p values. ( B ) HeLa cell (left) and HeLa cell overexpressing Centrobin:GFP (right), immunostained for GFP and CP110. ( C ) Confocal images of HeLa cells overexpressing TRIM37:GFP immunostained with antibodies against GFP and Centrobin. Source data for panel A can be found in .

Techniques: Real-time Polymerase Chain Reaction

( A ) Control and patient-2 fibroblasts immunostained for Centrin-2 and Centrobin. ( B ) Corresponding percentage of cells bearing Centrobin structures. Chart shows the average and SDs from three independent experiments (n: total number of cells scored per condition). Here and in other charts of this figure, two conditions that do not share the same letter are significantly different from each other, with p<0.05; unpaired Student’s t-test; see for exact p values. ( C ) Control and patient-2 fibroblasts in mitosis immunostained for Centrin-2, Centrobin and α-tubulin. Note that Centrobin structures can either act as MTOCs (inset 3, top cell) or not (inset 3, bottom cell). ( D ) Corresponding percentage of metaphase cells with Centrobin structures either associated or not associated to an active MTOC. Chart shows the average and SDs from three independent experiments (n: total number of cells scored per condition). Lower-case and upper-case letters above the charts reflect comparisons of two distinct data sets. ( E ) HeLa cells were synchronized with a double thymidine block, released and transfected with TRIM37 siRNAs 24 hr before second thymidine release. Cells were fixed and immunostained with antibodies against Centrin-2 and Centrobin at the time of transfection (−24 hr) and at the indicated times after release. ( F ) Corresponding percentage of cells with Centrobin assemblies either in close proximity to Cenpas or else not associated with them. Chart shows the average and SDs from three independent experiments (n = 50 cells each). ( G ) Control and Centrobin-ko RPE-1 cells transfected with TRIM37 siRNAs immunostained for Centrin-2 and CP110. ( H ) Corresponding percentages of mitotic cells with >4 CP110 foci. Chart shows the average and SDs from three independent experiments (n = 50 cells each). Source data for panels B, D, F, and H can be found in . Figure 5—source data 1. Source data for figure panels: , .

Journal: eLife

Article Title: TRIM37 prevents formation of centriolar protein assemblies by regulating Centrobin

doi: 10.7554/eLife.62640

Figure Lengend Snippet: ( A ) Control and patient-2 fibroblasts immunostained for Centrin-2 and Centrobin. ( B ) Corresponding percentage of cells bearing Centrobin structures. Chart shows the average and SDs from three independent experiments (n: total number of cells scored per condition). Here and in other charts of this figure, two conditions that do not share the same letter are significantly different from each other, with p<0.05; unpaired Student’s t-test; see for exact p values. ( C ) Control and patient-2 fibroblasts in mitosis immunostained for Centrin-2, Centrobin and α-tubulin. Note that Centrobin structures can either act as MTOCs (inset 3, top cell) or not (inset 3, bottom cell). ( D ) Corresponding percentage of metaphase cells with Centrobin structures either associated or not associated to an active MTOC. Chart shows the average and SDs from three independent experiments (n: total number of cells scored per condition). Lower-case and upper-case letters above the charts reflect comparisons of two distinct data sets. ( E ) HeLa cells were synchronized with a double thymidine block, released and transfected with TRIM37 siRNAs 24 hr before second thymidine release. Cells were fixed and immunostained with antibodies against Centrin-2 and Centrobin at the time of transfection (−24 hr) and at the indicated times after release. ( F ) Corresponding percentage of cells with Centrobin assemblies either in close proximity to Cenpas or else not associated with them. Chart shows the average and SDs from three independent experiments (n = 50 cells each). ( G ) Control and Centrobin-ko RPE-1 cells transfected with TRIM37 siRNAs immunostained for Centrin-2 and CP110. ( H ) Corresponding percentages of mitotic cells with >4 CP110 foci. Chart shows the average and SDs from three independent experiments (n = 50 cells each). Source data for panels B, D, F, and H can be found in . Figure 5—source data 1. Source data for figure panels: , .

Techniques: Blocking Assay, Transfection

$( A ) HeLa cells in G2 or mitosis, as indicated, treated with control or Centrobin siRNAs, and immunostained for Centrobin plus CP110. ( B ) Corresponding percentage of mitotic cells with indicated number of CP110 foci. In the statistical analysis, lower-case, upper-case and italic letters indicate three independent set of data analyzed. Chart shows the average and SDs from three independent experiments (n = 50 cells each). Here and in other charts of this figure, two conditions that do not share the same letter are significantly different from each other, with p<0.05; unpaired Student’s t-test; see for exact p values. ( C ) HeLa cells depleted of TRIM37 alone or simultaneously of TRIM37 and Centrobin, and immunostained for CP110 plus Centrobin. ( D ) Corresponding percentages of mitotic cells treated with the indicated siRNAs harboring >4 CP110 foci. Chart shows the average and SDs from two independent experiments (n = 50 cells each). ( E ) Western blot of HeLa cells depleted of TRIM37 or Centrobin alone and simultaneously of TRIM37 and Centrobin. probed with antibodies against Centrobin (top), TRIM37 (middle), or α-tubulin as loading control (bottom). ( F ) Western blot of soluble (S) and insoluble (P, for pellet) fractions of lysates from HeLa cells transfected with siRNAs against TRIM37 or against both TRIM37 and Centrobin, probed with antibodies against Centrobin (top) or α-tubulin as loading control (bottom). ( G ) Control or Centrobin-ko RPE-1 cells transfected with control siRNAs and immunostained for Centrin-2 plus CP110. Source data for panels B and D can be found in .$

Journal: eLife

Article Title: TRIM37 prevents formation of centriolar protein assemblies by regulating Centrobin

doi: 10.7554/eLife.62640

Figure Lengend Snippet: ( A ) HeLa cells in G2 or mitosis, as indicated, treated with control or Centrobin siRNAs, and immunostained for Centrobin plus CP110. ( B ) Corresponding percentage of mitotic cells with indicated number of CP110 foci. In the statistical analysis, lower-case, upper-case and italic letters indicate three independent set of data analyzed. Chart shows the average and SDs from three independent experiments (n = 50 cells each). Here and in other charts of this figure, two conditions that do not share the same letter are significantly different from each other, with p<0.05; unpaired Student’s t-test; see for exact p values. ( C ) HeLa cells depleted of TRIM37 alone or simultaneously of TRIM37 and Centrobin, and immunostained for CP110 plus Centrobin. ( D ) Corresponding percentages of mitotic cells treated with the indicated siRNAs harboring >4 CP110 foci. Chart shows the average and SDs from two independent experiments (n = 50 cells each). ( E ) Western blot of HeLa cells depleted of TRIM37 or Centrobin alone and simultaneously of TRIM37 and Centrobin. probed with antibodies against Centrobin (top), TRIM37 (middle), or α-tubulin as loading control (bottom). ( F ) Western blot of soluble (S) and insoluble (P, for pellet) fractions of lysates from HeLa cells transfected with siRNAs against TRIM37 or against both TRIM37 and Centrobin, probed with antibodies against Centrobin (top) or α-tubulin as loading control (bottom). ( G ) Control or Centrobin-ko RPE-1 cells transfected with control siRNAs and immunostained for Centrin-2 plus CP110. Source data for panels B and D can be found in .

Techniques: Western Blot, Transfection

$( A ) Box-and-whisker Tukey plot of Centrin-2 foci number per cell in indicated conditions. Box plots show median, interquartile range (10–90 percentile) and SDs from two independent experiments (n = 50 cells each). All cells were analyzed in mitosis with the exception of HsSAS-6-ko conditions. Here and in other charts of this figure, two conditions that do not share the same letter are significantly different from each other, with p<0.05; unpaired Student’s t-test; see for exact p values. Lower-case and upper-case letters above the charts reflect comparisons of two distinct data sets. ( B ) HeLa cells grown with centrinone for 8 days (top) or RPE-1 HsSAS-6-ko cells (bottom), both treated with control or TRIM37 siRNAs, before immunostaining for CP110 and Centrin-2. ( C–D ) HeLa cells grown with centrinone for 8 days ( C ) or RPE-1 HsSAS-6-ko cells ( D ), both treated with control or TRIM37 siRNAs, before immunostaining for Centrobin and Centrin-2. ( E ) HeLa cells were synchronized with a double thymidine block, released and transfected with control, TRIM37, Centrobin, or both TRIM37 and Centrobin siRNAs, as indicated. Additionally, DMSO or BI-2536 was added to the cells, which were fixed at time 0 hr or 8 hr after release, before immunostaining with antibodies against CP110 and Centrobin. The percentage of cells with extra CP110 foci was quantified in each condition. Chart shows the average and SDs from three independent experiments (n = 50 cells each). ( F ) HeLa cells were synchronized with a double thymidine block, released and transfected with control, TRIM37, Centrobin, or both TRIM37 and Centrobin siRNAs, as indicated. Moreover, DMSO or BI-2536 was added to the cells, which were fixed at time 0 hr or 8 hr after release, before immunostaining with antibodies against CP110 and HsSAS-6. ( G ) Corresponding percentage of cells with extra CP110 foci, with an indication of the fraction of them bearing HsSAS-6. Chart shows the average and SDs from two independent experiments (n = 50 cells each). ( H ) Working model of TRIM37 role in preventing formation of supernumerary MTOCs. Our findings lead us to propose that TRIM37 prevents the formation of supernumerary Centrin foci through two pathways mediated by Centrobin (top) and PLK1 (bottom). The Centrobin pathway relies on tiger Centrobin assemblies that act as platforms for PLK4-dependent Cenpas formation. Thereafter, Cenpas could evolve into centriole-related structures with the stepwise incorporation of other centriolar proteins such as HsSAS-6. We propose that the PLK1 pathway might reflect its role in promoting centriole disengagement. Note that only extra MTOCs are represented. See text for details. Source data for panels A, E, and G can be found in . Figure 6—source data 1. Source data for figure panels: and .$

Journal: eLife

Article Title: TRIM37 prevents formation of centriolar protein assemblies by regulating Centrobin

doi: 10.7554/eLife.62640

Figure Lengend Snippet: ( A ) Box-and-whisker Tukey plot of Centrin-2 foci number per cell in indicated conditions. Box plots show median, interquartile range (10–90 percentile) and SDs from two independent experiments (n = 50 cells each). All cells were analyzed in mitosis with the exception of HsSAS-6-ko conditions. Here and in other charts of this figure, two conditions that do not share the same letter are significantly different from each other, with p<0.05; unpaired Student’s t-test; see for exact p values. Lower-case and upper-case letters above the charts reflect comparisons of two distinct data sets. ( B ) HeLa cells grown with centrinone for 8 days (top) or RPE-1 HsSAS-6-ko cells (bottom), both treated with control or TRIM37 siRNAs, before immunostaining for CP110 and Centrin-2. ( C–D ) HeLa cells grown with centrinone for 8 days ( C ) or RPE-1 HsSAS-6-ko cells ( D ), both treated with control or TRIM37 siRNAs, before immunostaining for Centrobin and Centrin-2. ( E ) HeLa cells were synchronized with a double thymidine block, released and transfected with control, TRIM37, Centrobin, or both TRIM37 and Centrobin siRNAs, as indicated. Additionally, DMSO or BI-2536 was added to the cells, which were fixed at time 0 hr or 8 hr after release, before immunostaining with antibodies against CP110 and Centrobin. The percentage of cells with extra CP110 foci was quantified in each condition. Chart shows the average and SDs from three independent experiments (n = 50 cells each). ( F ) HeLa cells were synchronized with a double thymidine block, released and transfected with control, TRIM37, Centrobin, or both TRIM37 and Centrobin siRNAs, as indicated. Moreover, DMSO or BI-2536 was added to the cells, which were fixed at time 0 hr or 8 hr after release, before immunostaining with antibodies against CP110 and HsSAS-6. ( G ) Corresponding percentage of cells with extra CP110 foci, with an indication of the fraction of them bearing HsSAS-6. Chart shows the average and SDs from two independent experiments (n = 50 cells each). ( H ) Working model of TRIM37 role in preventing formation of supernumerary MTOCs. Our findings lead us to propose that TRIM37 prevents the formation of supernumerary Centrin foci through two pathways mediated by Centrobin (top) and PLK1 (bottom). The Centrobin pathway relies on tiger Centrobin assemblies that act as platforms for PLK4-dependent Cenpas formation. Thereafter, Cenpas could evolve into centriole-related structures with the stepwise incorporation of other centriolar proteins such as HsSAS-6. We propose that the PLK1 pathway might reflect its role in promoting centriole disengagement. Note that only extra MTOCs are represented. See text for details. Source data for panels A, E, and G can be found in . Figure 6—source data 1. Source data for figure panels: and .

Techniques: Whisker Assay, Immunostaining, Blocking Assay, Transfection

Journal: eLife

Article Title: TRIM37 prevents formation of centriolar protein assemblies by regulating Centrobin

doi: 10.7554/eLife.62640

Figure Lengend Snippet: ( A ) Percentages of cells with Centrobin structures in the indicated conditions. Chart shows the average and SDs at least from two independent experiments (n = 50 cells each). In the statistical analysis, lower-case and upper-case letters indicate two independent set of data analyzed. Two conditions that do not share the same letter are significantly different from each other, with p<0.05; unpaired Student’s t-test; see for exact p values. ( B ) FACs profile of HeLa cells synchronized with a double thymidine block, released and transfected with control, TRIM37, Centrobin, or both TRIM37 and Centrobin siRNAs, as indicated. Additionally, DMSO or BI-2536 was added to the cells, which were fixed at time 0 hr or 8 hr after release. Source data for panel A can be found in .

Techniques: Blocking Assay, Transfection

Journal: eLife

Article Title: TRIM37 prevents formation of centriolar protein assemblies by regulating Centrobin

doi: 10.7554/eLife.62640

Figure Lengend Snippet:

Techniques: Plasmid Preparation, Expressing, Mutagenesis, Recombinant, Sequencing, Negative Control

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1) Product Images from "TRIM37 Augments AP-2γ Transcriptional Activity and Cellular Localization via K63-linked Ubiquitination to Drive Breast Cancer Progression"

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