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( A ) The incidence rate of TNBC in female BW and White women (WW), excluding Hispanics, from 2010 to 2020 Surveillance, Epidemiology, and End Results (SEER) data. P value for years 2010–2020 *** P < 2.2 × 10 −16 , proportion test. ( B , C ) TNBC incidence ( B ) and mortality rate ( C ) per 100,000 for BW and WW TNBC patients stratified by age groups 15–39, 40–74, and above 75 years of age from 2010 to 2020. P values for the incidence and mortality rates in the different groups are ***<2.2 × 10 −16 , proportion test. ( D , E ) Box plot for racial identity-specific <t>TRIM37</t> expression in Stage I ( D ; n = 71) and Stage (II–IV; n = 248) TNBC patients (GSE142731, Data ref: Saleh et al, ; PRJNA704957 , Data ref: NCBI , , and TCGA, Data ref: . Stage I, * P = 0.029, and Stage II–IV, ns P = 0.058, unpaired t test. The boxed areas span the first to the third quartile, with the central line representing the median expression changes for each group. Outliers from the boxplots are not displayed. The whiskers represent the 15th and 85th percentiles. ( F ) Kaplan–Meier survival curve showing overall survival for the BW and WW TNBC patients with high TRIM37 expression (GSE39004; Data ref: Tang et al, ”, TCGA, Data ref: , and GSE18229, Data ref: Prat et al, ). ** P = 0.0011, log-rank test. The number of surviving patients at 0, 100, 200, and 300-month time points is indicated below the graph. ( G ) Box plot for TRIM37 expression in the normal breast tissue of BW ( n = 119) and WW ( n = 430) (GSE164641, Data ref: Marino et al, ; GSE111601, Data ref: Sun et al, ; GTEX, Data ref: , and TCGA, Data ref: . ** P = 0.0029, unpaired t test. The boxed areas span the first to the third quartile, with the central line representing the median expression changes for each group. Outliers from the boxplots are not displayed. The whiskers represent the 15th and 85th percentiles. ( H ) Association of TRIM37 expression in the cancer-free breast tissue with racial identity and age by chi-squared, univariate, and multivariate analyses. ( I ) Box plot of TRIM37 transcript level in the normal breast tissue of BW ( n = 6) and WW ( n = 103) ancestry confirmed by admixture analysis (TCGA, Data ref: ). * P = 0.049, Wilcoxon test. The boxed areas span the first to the third quartile, with the central line representing the median expression changes for each group. Outliers from the boxplots are not displayed. The whiskers represent the 15th and 85th percentiles. ( J ) Box plot for TRIM37 transcript levels in breast tissues from women at average risk ( n = 106) or high risk ( n = 72) of developing breast cancer (GSE164641, Data ref: Marino et al, ). ** P = 0.0022, unpaired t test. The boxed areas span the first to the third quartile, with the central line representing the median expression changes for each group. Outliers from the boxplots are not displayed. The whiskers represent the 15th and 85th percentiles. ( K ) Association of TRIM37 expression in breast tissues from women at average ( n = 106) or high risk ( n = 72) of developing breast cancer with clinicopathological variables (breast cancer risk, age, racial identity, BMI, menopausal status, and parity) by chi-squared, univariate, and multivariate analyses. .
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(A) A diagram depicting <t>TRIM37</t> dysregulation in MUL and 17q23-amplified cancers. In MUL, TRIM37 loss-of-function mutations result in the aberrant formation of Centrobin assemblies, which act as ectopic MTOCs during mitosis. Conversely, in 17q23-amplified cancers, elevated expression of TRIM37 leads to excessive degradation of centrosomal CEP192. (B) Centre, domain architecture of TRIM37 (UniProt ID 094972) highlighting the common RBCC motif (RING, B-box, Coiled-coil domains) and unique C-terminal TRAF domain. Surrounding panels, localization pattern and effect of HA-tagged TRIM37 variants (domain-specific mutations and deletions) on centrosomal CEP192 levels in RPE-1 tet-on TRIM37 cells. MUL indicates Mulibrey nanism patient-derived mutations. Representative data; n = 3 biological replicates. Scale bars, 5 μm. (C) Quantification of Centrobin assembly occurrence in RPE-1 TRIM37 -/- cells expressing the indicated HA-tagged TRIM37 variants. n = 3 biological replicates, each with >100 cells. P values, one-way ANOVA with post hoc Dunnett’s multiple comparisons test to evaluate differences between the TRIM37 variants and wild-type (WT). Mean ± s.e.m. (D) Quantification of centrosomal CEP192 signal in RPE-1 tet-on TRIM37 cells from (B). n = 3 biological replicates, each with >100 cells. P values, unpaired two-tailed t -test. Mean ± s.e.m. (E) Left, AlphaFold-predicted monomer of TRIM37. The RING, B-box, Coiled-coil, and TRAF domains are shown, with mutated residues highlighted in red. Right, AlphaFold Multimer-predicted model of a TRIM37 dimer. For both models, the unstructured C-terminal tail of TRIM37 (residues 449–964) is not shown due to the lack of a high-confidence prediction.
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A The mRNA and protein expressions of <t>TRIM37</t> in KIFC1-overexpressed HEC-1A and KIFC1-knockdown Ishikawa cells. B The mRNA and protein expressions of TRIM37 in PLK4-overexpressed and KIFC1-knockdown EC cell lines (HEC-1A and Ishikawa). C Results of the ubiquitination level of PLK4 in HEK293T cells after KIFC1 or TRIM37 overexpression detected by ubiquitination assay. D The mRNA expressions of KIFC1, TRIM37, and PLK4 in TRIM37- and KIFC1-overexpressed EC cell lines (HEC-1A and Ishikawa). E CCK8 assay in TRIM37- and KIFC1-overexpressed EC cell lines (HEC-1A and Ishikawa). F Colony formation assay in TRIM37- and KIFC1-overexpressed EC cell lines (HEC-1A and Ishikawa). G Transwell assay and quantitative analysis of migratory and invasive abilities of TRIM37- and KIFC1-overexpressed EC cell lines (HEC-1A and Ishikawa) (scale bar: 100 μm). H The protein expressions of KIFC1, TRIM37, and PLK4 in TRIM37- and KIFC1-overexpressed EC cell lines (HEC-1A and Ishikawa). * P < 0.05, ** P < 0.01, # P < 0.05, ## P < 0.01.
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A The mRNA and protein expressions of <t>TRIM37</t> in KIFC1-overexpressed HEC-1A and KIFC1-knockdown Ishikawa cells. B The mRNA and protein expressions of TRIM37 in PLK4-overexpressed and KIFC1-knockdown EC cell lines (HEC-1A and Ishikawa). C Results of the ubiquitination level of PLK4 in HEK293T cells after KIFC1 or TRIM37 overexpression detected by ubiquitination assay. D The mRNA expressions of KIFC1, TRIM37, and PLK4 in TRIM37- and KIFC1-overexpressed EC cell lines (HEC-1A and Ishikawa). E CCK8 assay in TRIM37- and KIFC1-overexpressed EC cell lines (HEC-1A and Ishikawa). F Colony formation assay in TRIM37- and KIFC1-overexpressed EC cell lines (HEC-1A and Ishikawa). G Transwell assay and quantitative analysis of migratory and invasive abilities of TRIM37- and KIFC1-overexpressed EC cell lines (HEC-1A and Ishikawa) (scale bar: 100 μm). H The protein expressions of KIFC1, TRIM37, and PLK4 in TRIM37- and KIFC1-overexpressed EC cell lines (HEC-1A and Ishikawa). * P < 0.05, ** P < 0.01, # P < 0.05, ## P < 0.01.
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A The mRNA and protein expressions of <t>TRIM37</t> in KIFC1-overexpressed HEC-1A and KIFC1-knockdown Ishikawa cells. B The mRNA and protein expressions of TRIM37 in PLK4-overexpressed and KIFC1-knockdown EC cell lines (HEC-1A and Ishikawa). C Results of the ubiquitination level of PLK4 in HEK293T cells after KIFC1 or TRIM37 overexpression detected by ubiquitination assay. D The mRNA expressions of KIFC1, TRIM37, and PLK4 in TRIM37- and KIFC1-overexpressed EC cell lines (HEC-1A and Ishikawa). E CCK8 assay in TRIM37- and KIFC1-overexpressed EC cell lines (HEC-1A and Ishikawa). F Colony formation assay in TRIM37- and KIFC1-overexpressed EC cell lines (HEC-1A and Ishikawa). G Transwell assay and quantitative analysis of migratory and invasive abilities of TRIM37- and KIFC1-overexpressed EC cell lines (HEC-1A and Ishikawa) (scale bar: 100 μm). H The protein expressions of KIFC1, TRIM37, and PLK4 in TRIM37- and KIFC1-overexpressed EC cell lines (HEC-1A and Ishikawa). * P < 0.05, ** P < 0.01, # P < 0.05, ## P < 0.01.
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( A ) The incidence rate of TNBC in female BW and White women (WW), excluding Hispanics, from 2010 to 2020 Surveillance, Epidemiology, and End Results (SEER) data. P value for years 2010–2020 *** P < 2.2 × 10 −16 , proportion test. ( B , C ) TNBC incidence ( B ) and mortality rate ( C ) per 100,000 for BW and WW TNBC patients stratified by age groups 15–39, 40–74, and above 75 years of age from 2010 to 2020. P values for the incidence and mortality rates in the different groups are ***<2.2 × 10 −16 , proportion test. ( D , E ) Box plot for racial identity-specific TRIM37 expression in Stage I ( D ; n = 71) and Stage (II–IV; n = 248) TNBC patients (GSE142731, Data ref: Saleh et al, ; PRJNA704957 , Data ref: NCBI , , and TCGA, Data ref: . Stage I, * P = 0.029, and Stage II–IV, ns P = 0.058, unpaired t test. The boxed areas span the first to the third quartile, with the central line representing the median expression changes for each group. Outliers from the boxplots are not displayed. The whiskers represent the 15th and 85th percentiles. ( F ) Kaplan–Meier survival curve showing overall survival for the BW and WW TNBC patients with high TRIM37 expression (GSE39004; Data ref: Tang et al, ”, TCGA, Data ref: , and GSE18229, Data ref: Prat et al, ). ** P = 0.0011, log-rank test. The number of surviving patients at 0, 100, 200, and 300-month time points is indicated below the graph. ( G ) Box plot for TRIM37 expression in the normal breast tissue of BW ( n = 119) and WW ( n = 430) (GSE164641, Data ref: Marino et al, ; GSE111601, Data ref: Sun et al, ; GTEX, Data ref: , and TCGA, Data ref: . ** P = 0.0029, unpaired t test. The boxed areas span the first to the third quartile, with the central line representing the median expression changes for each group. Outliers from the boxplots are not displayed. The whiskers represent the 15th and 85th percentiles. ( H ) Association of TRIM37 expression in the cancer-free breast tissue with racial identity and age by chi-squared, univariate, and multivariate analyses. ( I ) Box plot of TRIM37 transcript level in the normal breast tissue of BW ( n = 6) and WW ( n = 103) ancestry confirmed by admixture analysis (TCGA, Data ref: ). * P = 0.049, Wilcoxon test. The boxed areas span the first to the third quartile, with the central line representing the median expression changes for each group. Outliers from the boxplots are not displayed. The whiskers represent the 15th and 85th percentiles. ( J ) Box plot for TRIM37 transcript levels in breast tissues from women at average risk ( n = 106) or high risk ( n = 72) of developing breast cancer (GSE164641, Data ref: Marino et al, ). ** P = 0.0022, unpaired t test. The boxed areas span the first to the third quartile, with the central line representing the median expression changes for each group. Outliers from the boxplots are not displayed. The whiskers represent the 15th and 85th percentiles. ( K ) Association of TRIM37 expression in breast tissues from women at average ( n = 106) or high risk ( n = 72) of developing breast cancer with clinicopathological variables (breast cancer risk, age, racial identity, BMI, menopausal status, and parity) by chi-squared, univariate, and multivariate analyses. .

Journal: EMBO Reports

Article Title: The TRIM37 variant rs57141087 contributes to triple-negative breast cancer outcomes in Black women

doi: 10.1038/s44319-024-00331-2

Figure Lengend Snippet: ( A ) The incidence rate of TNBC in female BW and White women (WW), excluding Hispanics, from 2010 to 2020 Surveillance, Epidemiology, and End Results (SEER) data. P value for years 2010–2020 *** P < 2.2 × 10 −16 , proportion test. ( B , C ) TNBC incidence ( B ) and mortality rate ( C ) per 100,000 for BW and WW TNBC patients stratified by age groups 15–39, 40–74, and above 75 years of age from 2010 to 2020. P values for the incidence and mortality rates in the different groups are ***<2.2 × 10 −16 , proportion test. ( D , E ) Box plot for racial identity-specific TRIM37 expression in Stage I ( D ; n = 71) and Stage (II–IV; n = 248) TNBC patients (GSE142731, Data ref: Saleh et al, ; PRJNA704957 , Data ref: NCBI , , and TCGA, Data ref: . Stage I, * P = 0.029, and Stage II–IV, ns P = 0.058, unpaired t test. The boxed areas span the first to the third quartile, with the central line representing the median expression changes for each group. Outliers from the boxplots are not displayed. The whiskers represent the 15th and 85th percentiles. ( F ) Kaplan–Meier survival curve showing overall survival for the BW and WW TNBC patients with high TRIM37 expression (GSE39004; Data ref: Tang et al, ”, TCGA, Data ref: , and GSE18229, Data ref: Prat et al, ). ** P = 0.0011, log-rank test. The number of surviving patients at 0, 100, 200, and 300-month time points is indicated below the graph. ( G ) Box plot for TRIM37 expression in the normal breast tissue of BW ( n = 119) and WW ( n = 430) (GSE164641, Data ref: Marino et al, ; GSE111601, Data ref: Sun et al, ; GTEX, Data ref: , and TCGA, Data ref: . ** P = 0.0029, unpaired t test. The boxed areas span the first to the third quartile, with the central line representing the median expression changes for each group. Outliers from the boxplots are not displayed. The whiskers represent the 15th and 85th percentiles. ( H ) Association of TRIM37 expression in the cancer-free breast tissue with racial identity and age by chi-squared, univariate, and multivariate analyses. ( I ) Box plot of TRIM37 transcript level in the normal breast tissue of BW ( n = 6) and WW ( n = 103) ancestry confirmed by admixture analysis (TCGA, Data ref: ). * P = 0.049, Wilcoxon test. The boxed areas span the first to the third quartile, with the central line representing the median expression changes for each group. Outliers from the boxplots are not displayed. The whiskers represent the 15th and 85th percentiles. ( J ) Box plot for TRIM37 transcript levels in breast tissues from women at average risk ( n = 106) or high risk ( n = 72) of developing breast cancer (GSE164641, Data ref: Marino et al, ). ** P = 0.0022, unpaired t test. The boxed areas span the first to the third quartile, with the central line representing the median expression changes for each group. Outliers from the boxplots are not displayed. The whiskers represent the 15th and 85th percentiles. ( K ) Association of TRIM37 expression in breast tissues from women at average ( n = 106) or high risk ( n = 72) of developing breast cancer with clinicopathological variables (breast cancer risk, age, racial identity, BMI, menopausal status, and parity) by chi-squared, univariate, and multivariate analyses. .

Article Snippet: Immunoblots were probed using antibodies against Gapdh (Thermofisher), TRIM37 (Cell Signaling), and RAS (Cell Signaling).

Techniques: Expressing

( A ) Breast cancer incidence rate per 100,000 in women, excluding Hispanics, from 2015-2020 Surveillance, Epidemiology, and End Results (SEER) data. * P = 0.038, proportional test. The number of samples is indicated. ( B ) Univariate analyses of relationships between TRIM37 and racial identity in Stage I ( n = 71) and Stage II–IV ( n = 248) TNBC patients ( GSE142731 , Data ref: Saleh et al, ; PRJNA704957 , Data ref: NCBI , , and TCGA, Data ref: ) ( C ) Kaplan–Meier survival curve showing overall survival for the BW and WW TNBC patients with low TRIM37 expression ( GSE39004 , Data ref: Tang et al, ; TCGA, Data ref: , and GSE18229 , Data ref: Prat et al, ). The number of surviving patients at 0, 50, 100, 150, 200, and 250-month time points is indicated below the graph. ns P = 0.11, log-rank test. The number of surviving patients at 0, 50, 100, 150, 200, and 250-month time points are indicated. ( D ) Box plot for TRIM37 expression in the normal breast tissue by age, below or equal to 35 ( n = 115) vs. greater than 35 years ( n = 434) ( GSE164641 , Data ref: Marino et al, ”; GSE111601 , Data ref: Sun et al, ; GTEX, Data ref: , and TCGA, Data ref: ). ns P = 0.95, t test. The boxed areas span the first to the third quartile, with the central line representing the median expression changes for each group. Outliers from the boxplots are not displayed. The whiskers represent the 15th and 85th percentiles. ( E ) Estimated genetic ancestry distribution for self-reported BW ( n = 6) and WW ( n = 103) in the TCGA cohort (Data ref: ). Each column represents an individual in the cohort, and the estimated proportion of African, European, and Asian ancestry is shown on the y-axis. ( F – J ) Box plot for TRIM37 expression in the breast tissue from women stratified by racial identity (Black vs. White), parity (No vs. yes), menopausal status (Pre-vs. Post), age (below or equal to 35 vs. greater than 35 years) and BMI (Healthy vs. overweight vs. obese) ( GSE164641 , Data ref: Marino et al, ). For race, * P = 0.05, parity, ns P = 0.087, menopausal status, ns P = 0.52, age, ns P = 0.3, and BMI, ns P = 0.17, t test. The number of samples is indicated. The boxed areas span the first to the third quartile, with the central line representing the median expression changes for each group. Outliers from the boxplots are not displayed. The whiskers represent the 15th and 85th percentiles.

Journal: EMBO Reports

Article Title: The TRIM37 variant rs57141087 contributes to triple-negative breast cancer outcomes in Black women

doi: 10.1038/s44319-024-00331-2

Figure Lengend Snippet: ( A ) Breast cancer incidence rate per 100,000 in women, excluding Hispanics, from 2015-2020 Surveillance, Epidemiology, and End Results (SEER) data. * P = 0.038, proportional test. The number of samples is indicated. ( B ) Univariate analyses of relationships between TRIM37 and racial identity in Stage I ( n = 71) and Stage II–IV ( n = 248) TNBC patients ( GSE142731 , Data ref: Saleh et al, ; PRJNA704957 , Data ref: NCBI , , and TCGA, Data ref: ) ( C ) Kaplan–Meier survival curve showing overall survival for the BW and WW TNBC patients with low TRIM37 expression ( GSE39004 , Data ref: Tang et al, ; TCGA, Data ref: , and GSE18229 , Data ref: Prat et al, ). The number of surviving patients at 0, 50, 100, 150, 200, and 250-month time points is indicated below the graph. ns P = 0.11, log-rank test. The number of surviving patients at 0, 50, 100, 150, 200, and 250-month time points are indicated. ( D ) Box plot for TRIM37 expression in the normal breast tissue by age, below or equal to 35 ( n = 115) vs. greater than 35 years ( n = 434) ( GSE164641 , Data ref: Marino et al, ”; GSE111601 , Data ref: Sun et al, ; GTEX, Data ref: , and TCGA, Data ref: ). ns P = 0.95, t test. The boxed areas span the first to the third quartile, with the central line representing the median expression changes for each group. Outliers from the boxplots are not displayed. The whiskers represent the 15th and 85th percentiles. ( E ) Estimated genetic ancestry distribution for self-reported BW ( n = 6) and WW ( n = 103) in the TCGA cohort (Data ref: ). Each column represents an individual in the cohort, and the estimated proportion of African, European, and Asian ancestry is shown on the y-axis. ( F – J ) Box plot for TRIM37 expression in the breast tissue from women stratified by racial identity (Black vs. White), parity (No vs. yes), menopausal status (Pre-vs. Post), age (below or equal to 35 vs. greater than 35 years) and BMI (Healthy vs. overweight vs. obese) ( GSE164641 , Data ref: Marino et al, ). For race, * P = 0.05, parity, ns P = 0.087, menopausal status, ns P = 0.52, age, ns P = 0.3, and BMI, ns P = 0.17, t test. The number of samples is indicated. The boxed areas span the first to the third quartile, with the central line representing the median expression changes for each group. Outliers from the boxplots are not displayed. The whiskers represent the 15th and 85th percentiles.

Article Snippet: Immunoblots were probed using antibodies against Gapdh (Thermofisher), TRIM37 (Cell Signaling), and RAS (Cell Signaling).

Techniques: Expressing

( A ) Volcano plot illustrates differential gene expression in the cancer-free paraffin-archived breast tissue samples of parous and premenopausal and WW women ( n = 3 biological replicates; ages <45). Differences in gene expression between BW and WW were tested using DESeq2 from Bioconductor. FDR < 0.1. Red are significantly upregulated genes ( n = 2714), blue are significantly downregulated genes ( n = 2239), and grey are genes not significantly changed ( n = 11,448). ( B ) Venn diagram showing the overlap between differentially expressed genes in breast tissue from BW identified in ( A ) and TRIM37 -regulated genes (GSE136617, Data ref: Przanowski et al, ). ( C ) Hierarchical clustering of median-centered TRIM37 -TS in cancer-free BW and WW breast tissue ( n = 3 biological replicates per group). Each colored line in the dendrogram identifies a different gene. ( D ) Enrichment plots for cancer stem cells (top) and EMT (bottom) gene signatures enriched in cancer-free BW ( n = 3 biological replicates) breast tissue identified through GSEA analysis of RNA-seq data. Nominal P values were computed using the t test. ( E ) Kaplan–Meier analysis of survival in patients with low ( black ) or high ( red ) TRIM37 -regulated CSC (top) and EMT (bottom) gene signatures enriched in cancer-free BW ( n = 3 biological replicates) breast tissue identified in ( B ). For CSC signatures, * P = 0.033; for EMT signatures, * P = 0.031, log-rank test. The number of surviving patients at 0, 50, 100, and 150-month time points is indicated below the graph. ( F ) Volcano plot illustrates differential gene expression in control and TRIM37 overexpressing KTB51 breast epithelial cells ( n = 2 biological replicates per group). Differential gene expression between the groups was analyzed using DESeq2 from Bioconductor. FDR < 0.05. Red are significantly upregulated genes ( n = 3213), blue are significantly downregulated genes ( n = 3520), and grey are genes not significantly changed ( n = 7113). ( G ) Enrichment plots for cancer stem cells (left) and EMT (right) gene signatures identified through GSEA analysis of RNA-seq data for TRIM37 overexpressing KTB51 cells. Nominal P values were computed using a t test. ( H ) Volcano plot illustrates differential gene expression in control ( n = 2 biological replicates) and TRIM37 overexpressing ( n = 2 biological replicates) KTB39 breast epithelial cells. Differential gene expression between the groups was analyzed using DESeq2 from Bioconductor. FDR < 0.05. Red are significantly upregulated genes ( n = 2560), blue are significantly downregulated genes ( n = 3210) and grey are genes not significantly changed ( n = 7769). ( I ) Enrichment plots for cancer stem cells (left) and EMT (right) gene signatures identified through GSEA analysis of RNA-seq data for TRIM37 overexpressing KTB39 cells. Nominal P values were computed using a t test.

Journal: EMBO Reports

Article Title: The TRIM37 variant rs57141087 contributes to triple-negative breast cancer outcomes in Black women

doi: 10.1038/s44319-024-00331-2

Figure Lengend Snippet: ( A ) Volcano plot illustrates differential gene expression in the cancer-free paraffin-archived breast tissue samples of parous and premenopausal and WW women ( n = 3 biological replicates; ages <45). Differences in gene expression between BW and WW were tested using DESeq2 from Bioconductor. FDR < 0.1. Red are significantly upregulated genes ( n = 2714), blue are significantly downregulated genes ( n = 2239), and grey are genes not significantly changed ( n = 11,448). ( B ) Venn diagram showing the overlap between differentially expressed genes in breast tissue from BW identified in ( A ) and TRIM37 -regulated genes (GSE136617, Data ref: Przanowski et al, ). ( C ) Hierarchical clustering of median-centered TRIM37 -TS in cancer-free BW and WW breast tissue ( n = 3 biological replicates per group). Each colored line in the dendrogram identifies a different gene. ( D ) Enrichment plots for cancer stem cells (top) and EMT (bottom) gene signatures enriched in cancer-free BW ( n = 3 biological replicates) breast tissue identified through GSEA analysis of RNA-seq data. Nominal P values were computed using the t test. ( E ) Kaplan–Meier analysis of survival in patients with low ( black ) or high ( red ) TRIM37 -regulated CSC (top) and EMT (bottom) gene signatures enriched in cancer-free BW ( n = 3 biological replicates) breast tissue identified in ( B ). For CSC signatures, * P = 0.033; for EMT signatures, * P = 0.031, log-rank test. The number of surviving patients at 0, 50, 100, and 150-month time points is indicated below the graph. ( F ) Volcano plot illustrates differential gene expression in control and TRIM37 overexpressing KTB51 breast epithelial cells ( n = 2 biological replicates per group). Differential gene expression between the groups was analyzed using DESeq2 from Bioconductor. FDR < 0.05. Red are significantly upregulated genes ( n = 3213), blue are significantly downregulated genes ( n = 3520), and grey are genes not significantly changed ( n = 7113). ( G ) Enrichment plots for cancer stem cells (left) and EMT (right) gene signatures identified through GSEA analysis of RNA-seq data for TRIM37 overexpressing KTB51 cells. Nominal P values were computed using a t test. ( H ) Volcano plot illustrates differential gene expression in control ( n = 2 biological replicates) and TRIM37 overexpressing ( n = 2 biological replicates) KTB39 breast epithelial cells. Differential gene expression between the groups was analyzed using DESeq2 from Bioconductor. FDR < 0.05. Red are significantly upregulated genes ( n = 2560), blue are significantly downregulated genes ( n = 3210) and grey are genes not significantly changed ( n = 7769). ( I ) Enrichment plots for cancer stem cells (left) and EMT (right) gene signatures identified through GSEA analysis of RNA-seq data for TRIM37 overexpressing KTB39 cells. Nominal P values were computed using a t test.

Article Snippet: Immunoblots were probed using antibodies against Gapdh (Thermofisher), TRIM37 (Cell Signaling), and RAS (Cell Signaling).

Techniques: Expressing, RNA Sequencing Assay, Control

( A ) Venn diagram showing the overlap between DEGs in the cancer-free breast tissue samples of parous and premenopausal BW ( n = 3; ages <45) and WW ( n = 3; ages <45). ( B ) The top ten pathways enriched in the cancer-free breast tissue samples of BW relative to WW identified by GSEA are shown. ( C ) Immunoblot analysis of control and TRIM37 overexpressing KTB39 and KTB51 cells. Two clones for each cell line are shown. Gapdh was used as a loading control. ( D ) Venn diagram showing the overlap between differentially expressed genes in control and TRIM37 overexpressing KTB51 cells. ( E ) The top ten pathways significantly enriched in TRIM37 overexpressing KTB51 cells relative to control cells identified by GSEA are shown. ( F ) Venn diagram showing the overlap between differentially expressed genes in control and TRIM37 overexpressing KTB39 cells. ( G ) The top ten pathways enriched in TRIM37 overexpressing KTB39 cells relative to control cells identified by GSEA are shown.

Journal: EMBO Reports

Article Title: The TRIM37 variant rs57141087 contributes to triple-negative breast cancer outcomes in Black women

doi: 10.1038/s44319-024-00331-2

Figure Lengend Snippet: ( A ) Venn diagram showing the overlap between DEGs in the cancer-free breast tissue samples of parous and premenopausal BW ( n = 3; ages <45) and WW ( n = 3; ages <45). ( B ) The top ten pathways enriched in the cancer-free breast tissue samples of BW relative to WW identified by GSEA are shown. ( C ) Immunoblot analysis of control and TRIM37 overexpressing KTB39 and KTB51 cells. Two clones for each cell line are shown. Gapdh was used as a loading control. ( D ) Venn diagram showing the overlap between differentially expressed genes in control and TRIM37 overexpressing KTB51 cells. ( E ) The top ten pathways significantly enriched in TRIM37 overexpressing KTB51 cells relative to control cells identified by GSEA are shown. ( F ) Venn diagram showing the overlap between differentially expressed genes in control and TRIM37 overexpressing KTB39 cells. ( G ) The top ten pathways enriched in TRIM37 overexpressing KTB39 cells relative to control cells identified by GSEA are shown.

Article Snippet: Immunoblots were probed using antibodies against Gapdh (Thermofisher), TRIM37 (Cell Signaling), and RAS (Cell Signaling).

Techniques: Western Blot, Control, Clone Assay

( A ) Distribution of SNPs across the TRIM37 gene (Chr17:59,108,921–58,982,138 Mb) and their association with TRIM37 expression in the breast tissue from GTEx. ( B ) Linkage disequilibrium ( r 2 ) heatmap of SNPs in TRIM37 gene in African (Yoruba, Luhya, Gambia, Mende, Esan, Americans of African Ancestry, African Caribbean) population from the 1000 Genome project data. ( C ) Luciferase reporter assays measure the promoter performance of the TRIM37 minimal promoter (Chr17: 59,107,052–59,106,707) in HEK293T, MCF10a, and MDA MB 231 cells. For HEK293T, *** P = 6.05*10 -5 , MCF10a, ** P = 0.008, and MDA MB 231, * P = 0.012, unpaired t test. Data are mean±SD of biological replicates, n = 3/group. ( D ) TNBC risk association with the risk allele of rs57141087 using GWAS data for African ancestry (Jia et al, ). ( E ) Luciferase reporter assays measuring the enhancer activity of DNA fragments (Chr17: 59,107,961–59,107,053) in HEK293T cells. The data is normalized to the vector only set to 1 and presented as a fold change in luminescence. of the different fragments in HEK293T. For 353 bp, *** P = 0.0001, 909 bp, * P = 0.012, and 353 bp vs. 909 bp, P = 0.539, unpaired t test. Data are mean±SD of biological replicates, n = 3/group.

Journal: EMBO Reports

Article Title: The TRIM37 variant rs57141087 contributes to triple-negative breast cancer outcomes in Black women

doi: 10.1038/s44319-024-00331-2

Figure Lengend Snippet: ( A ) Distribution of SNPs across the TRIM37 gene (Chr17:59,108,921–58,982,138 Mb) and their association with TRIM37 expression in the breast tissue from GTEx. ( B ) Linkage disequilibrium ( r 2 ) heatmap of SNPs in TRIM37 gene in African (Yoruba, Luhya, Gambia, Mende, Esan, Americans of African Ancestry, African Caribbean) population from the 1000 Genome project data. ( C ) Luciferase reporter assays measure the promoter performance of the TRIM37 minimal promoter (Chr17: 59,107,052–59,106,707) in HEK293T, MCF10a, and MDA MB 231 cells. For HEK293T, *** P = 6.05*10 -5 , MCF10a, ** P = 0.008, and MDA MB 231, * P = 0.012, unpaired t test. Data are mean±SD of biological replicates, n = 3/group. ( D ) TNBC risk association with the risk allele of rs57141087 using GWAS data for African ancestry (Jia et al, ). ( E ) Luciferase reporter assays measuring the enhancer activity of DNA fragments (Chr17: 59,107,961–59,107,053) in HEK293T cells. The data is normalized to the vector only set to 1 and presented as a fold change in luminescence. of the different fragments in HEK293T. For 353 bp, *** P = 0.0001, 909 bp, * P = 0.012, and 353 bp vs. 909 bp, P = 0.539, unpaired t test. Data are mean±SD of biological replicates, n = 3/group.

Article Snippet: Immunoblots were probed using antibodies against Gapdh (Thermofisher), TRIM37 (Cell Signaling), and RAS (Cell Signaling).

Techniques: Expressing, Luciferase, Activity Assay, Plasmid Preparation

( A , B ) A schematic view of the (Chr17: 59,107,405-59,106,446) region ( A ) and pie charts ( B ) showing the distribution of risk (red) and reference (black) alleles in BW and WW from the NCBI SNP database. The percentage of each haplotype in Black and White populations is indicated. ( C ) Luciferase reporter assays measure the promoter performance of the promoter harboring risk alleles for different SNPs (top) in HEK293T, MCF10a, and MDA MB 231 cells. The data are normalized to the reference allele and presented as a fold change in luminescence. For HEK293T; rs150880035 ns P = 0.463, rs57141087, ** P = 0.005, rs1029035382, ns P = 0.268, rs914034546, ** P = 0.007, rs1359804461, *** P = 0.0003, rs904164828, ns P = 0.621, rs572032837, ns P = 0.398, rs568274755, ns P = 0.383, rs1042165062, ***p = 0.001; For MCF10a rs150880035, ns P = 0.133, rs57141087, *** P = 9.4 × 10 −5 , rs1029035382, * P = 0.021, rs914034546, *** P = 7.72 × 10 −3 , rs1359804461, ns P = 0.330, rs904164828, ** P = 0.005, rs572032837, ** P = 0.005, rs568274755, * P = 0.045, rs1042165062, ns P = 0.053; For MDA MB 231; rs150880035, ns P = 0.731, rs57141087, ** P = 0.002, rs1029035382, ns P = 0.974, rs914034546, ** P = 0.007, rs1359804461, * P = 0.036, rs904164828, ns P = 0.974, rs572032837, ns P = 0.469, rs568274755, ns P = 0.928, rs1042165062, ** P = 0.006, unpaired t test. Data are mean ± SD of biological replicates, n ≥3/group. ( D ) rs57141087 eQTL analysis of genes within 126,784-bp (Chr17:59,108,921–58,982,138) window using data for breast tissue from GTEx. ( E ) eQTL analysis demonstrating the correlation between rs57141087 genotype and TRIM37 expression in samples from 1000 genome RNA-seq dataset ( n = 175, Yoruba and UTAH population, Data ref: Lappalainen et al, ). * P = 0.041, t test. The boxed areas span the first to the third quartile, with the central line representing the median expression changes for each group. Outliers from the boxplots are not displayed. The whiskers represent the 15th and 85th percentiles. ( F ) The LocusZoom plot of LD for rs57141087 and its LD-SNPs in African (Yoruba, Luhya, Gambia, Mende, Esan, Americans of African Ancestry, and African Caribbean) population from the 1000 Genome project data. ( G ) The epigenetic heatmap for H3K4me1, H3K27Ac, DHSs, and H3K4me3 for the (Chr17:59,108,921–58,982,138) region harboring rs57141087. The data was derived from the Cistrome Data Browser. ( H ) Luciferase reporter assays measuring the enhancer activity of DNA fragment (Chr17: 59,107,961–59,107,053) harboring risk alleles for rs57141087 in HEK293T cells. The data are normalized to the vector only set to 1 and presented as a fold change in luminescence. For 353 bp, *** P = 4.8*10 −4 , 909 bp, * P = 0.015, 353 bp vs. 909 bp, ns P = 0.418, unpaired t test. Data are mean±SD of biological replicates, n = 3/group. ( I ) Looping assay measures the promoter–enhancer interactions for in vitro synthesized DNA fragments (Chr17: 59,107,405-59,106,446) with risk or reference alleles. For Nuclear extract, ** P = 0.003, BSA, ns P = 0.947, ns DNase, P = 0.965, unpaired t test. Data are mean±SD of biological replicates, n = 3/group. .

Journal: EMBO Reports

Article Title: The TRIM37 variant rs57141087 contributes to triple-negative breast cancer outcomes in Black women

doi: 10.1038/s44319-024-00331-2

Figure Lengend Snippet: ( A , B ) A schematic view of the (Chr17: 59,107,405-59,106,446) region ( A ) and pie charts ( B ) showing the distribution of risk (red) and reference (black) alleles in BW and WW from the NCBI SNP database. The percentage of each haplotype in Black and White populations is indicated. ( C ) Luciferase reporter assays measure the promoter performance of the promoter harboring risk alleles for different SNPs (top) in HEK293T, MCF10a, and MDA MB 231 cells. The data are normalized to the reference allele and presented as a fold change in luminescence. For HEK293T; rs150880035 ns P = 0.463, rs57141087, ** P = 0.005, rs1029035382, ns P = 0.268, rs914034546, ** P = 0.007, rs1359804461, *** P = 0.0003, rs904164828, ns P = 0.621, rs572032837, ns P = 0.398, rs568274755, ns P = 0.383, rs1042165062, ***p = 0.001; For MCF10a rs150880035, ns P = 0.133, rs57141087, *** P = 9.4 × 10 −5 , rs1029035382, * P = 0.021, rs914034546, *** P = 7.72 × 10 −3 , rs1359804461, ns P = 0.330, rs904164828, ** P = 0.005, rs572032837, ** P = 0.005, rs568274755, * P = 0.045, rs1042165062, ns P = 0.053; For MDA MB 231; rs150880035, ns P = 0.731, rs57141087, ** P = 0.002, rs1029035382, ns P = 0.974, rs914034546, ** P = 0.007, rs1359804461, * P = 0.036, rs904164828, ns P = 0.974, rs572032837, ns P = 0.469, rs568274755, ns P = 0.928, rs1042165062, ** P = 0.006, unpaired t test. Data are mean ± SD of biological replicates, n ≥3/group. ( D ) rs57141087 eQTL analysis of genes within 126,784-bp (Chr17:59,108,921–58,982,138) window using data for breast tissue from GTEx. ( E ) eQTL analysis demonstrating the correlation between rs57141087 genotype and TRIM37 expression in samples from 1000 genome RNA-seq dataset ( n = 175, Yoruba and UTAH population, Data ref: Lappalainen et al, ). * P = 0.041, t test. The boxed areas span the first to the third quartile, with the central line representing the median expression changes for each group. Outliers from the boxplots are not displayed. The whiskers represent the 15th and 85th percentiles. ( F ) The LocusZoom plot of LD for rs57141087 and its LD-SNPs in African (Yoruba, Luhya, Gambia, Mende, Esan, Americans of African Ancestry, and African Caribbean) population from the 1000 Genome project data. ( G ) The epigenetic heatmap for H3K4me1, H3K27Ac, DHSs, and H3K4me3 for the (Chr17:59,108,921–58,982,138) region harboring rs57141087. The data was derived from the Cistrome Data Browser. ( H ) Luciferase reporter assays measuring the enhancer activity of DNA fragment (Chr17: 59,107,961–59,107,053) harboring risk alleles for rs57141087 in HEK293T cells. The data are normalized to the vector only set to 1 and presented as a fold change in luminescence. For 353 bp, *** P = 4.8*10 −4 , 909 bp, * P = 0.015, 353 bp vs. 909 bp, ns P = 0.418, unpaired t test. Data are mean±SD of biological replicates, n = 3/group. ( I ) Looping assay measures the promoter–enhancer interactions for in vitro synthesized DNA fragments (Chr17: 59,107,405-59,106,446) with risk or reference alleles. For Nuclear extract, ** P = 0.003, BSA, ns P = 0.947, ns DNase, P = 0.965, unpaired t test. Data are mean±SD of biological replicates, n = 3/group. .

Article Snippet: Immunoblots were probed using antibodies against Gapdh (Thermofisher), TRIM37 (Cell Signaling), and RAS (Cell Signaling).

Techniques: Luciferase, Expressing, RNA Sequencing Assay, Derivative Assay, Activity Assay, Plasmid Preparation, In Vitro, Synthesized

( A ) Representative polyacrylamide gel indicates a distinct HhaI digestion pattern for KTB51 clones harboring rs57141087 reference (A) and risk (G) alleles. The data for three different clones is shown. ( B ) Immunoblot for biotin-labeled TRIM37 enhancer fragment with risk (G allele) and reference (A allele) incubated with HEK293T nuclear extract and competitor (unlabeled probe) as indicated. The bound and free DNA fragments are indicated with red arrows, and the shift in the band is indicated (red asterisk, *). The concentrations of nuclear extract (5 μg), Biotin-labeled probes (0.2 pmol), competitor (10 pmol), and poly [d(I-C)] (50 ng/μl) were indicated at the Top . The representative images from two different experiments (Replicate #1–2) are shown. ( C , D ) The risk G allele of rs57141087 harboring GFI1 ( C ) and PAX5 ( D ) binding motif in the enhancer region of TRIM37 (Chr17: 59,107,267–59,107,262) is shown. ( E , F ) qRT-PCR monitoring TRIM37 levels in GFI1 (E) and PAX5 ( F ) knockdown in KTB51 cells with A or G allele for rs57141087. Gapdh is used as an endogenous control. For GFI1 knockdown, GFI1 (A), *** P = 0.001, TRIM37 (A), ns P = 0.831, GFI1 ( G ), * P = 0.024, and TRIM37 (G), ns P = 0.345. For PAX5 knockdown, PAX5 (A), *** P = 0.0005, TRIM37 (A), ns P = 0.187, PAX5 ( G ), *** P = 0.001, and TRIM37 ( G ), ns P = 0.874, unpaired t test. Data are mean±SD of biological replicates, n ≥3/group. ( G ) Immunoblot for biotin-labeled TRIM37 enhancer fragment incubated with nuclear extract from control or NRF1 knockdown HEK293T cells and unlabeled probe (competitor) as indicated. The bound and free DNA fragments are indicated with red arrows, and the shift in the band is indicated (red asterisk, *). The concentrations of nuclear extract (5 μg), Biotin-labeled probes (0.2 pmol), competitor (10 pmol), and poly [d(I-C)] (50 ng/μl) were indicated at the Top . The representative images from two different experiments (Replicate #1–2) are shown. ( H ) A looping assay measuring the promoter–enhancer interactions for in vitro synthesized DNA fragments (Chr17: 59,107,405-59,106,446) with risk or reference alleles or mutated NRF1 binding motifs in the promoter was used. For BSA, ns P = 0.947, NRF1, ** P = 0.003, NRF1 vs mutated NRF1, ** P = 0.009, and DNase, ns P = 0.965, unpaired t test. Data are mean±SD of biological replicates, n = 3/group. ( I ) Correlation plots of the FPKM read from RNA-seq analysis for each replicate are shown for control ( Left ) and 51-G ( Right ) cells. ( J ) Venn diagram showing the overlap between differentially expressed genes in control and 51-G cells. ( K ) qRT-PCR analysis monitoring TRIM37 in control and 51-G cells. Gapdh is used as an endogenous control. *** P = 0.0003, unpaired t test. Data are mean ± SD of biological replicates, n = 4/group. The boxed areas span the first to the third quartile. The whiskers represent the 15th and 85th percentiles. ( L ) The top ten pathways enriched in 51-G relative to control cells identified by GSEA are shown.

Journal: EMBO Reports

Article Title: The TRIM37 variant rs57141087 contributes to triple-negative breast cancer outcomes in Black women

doi: 10.1038/s44319-024-00331-2

Figure Lengend Snippet: ( A ) Representative polyacrylamide gel indicates a distinct HhaI digestion pattern for KTB51 clones harboring rs57141087 reference (A) and risk (G) alleles. The data for three different clones is shown. ( B ) Immunoblot for biotin-labeled TRIM37 enhancer fragment with risk (G allele) and reference (A allele) incubated with HEK293T nuclear extract and competitor (unlabeled probe) as indicated. The bound and free DNA fragments are indicated with red arrows, and the shift in the band is indicated (red asterisk, *). The concentrations of nuclear extract (5 μg), Biotin-labeled probes (0.2 pmol), competitor (10 pmol), and poly [d(I-C)] (50 ng/μl) were indicated at the Top . The representative images from two different experiments (Replicate #1–2) are shown. ( C , D ) The risk G allele of rs57141087 harboring GFI1 ( C ) and PAX5 ( D ) binding motif in the enhancer region of TRIM37 (Chr17: 59,107,267–59,107,262) is shown. ( E , F ) qRT-PCR monitoring TRIM37 levels in GFI1 (E) and PAX5 ( F ) knockdown in KTB51 cells with A or G allele for rs57141087. Gapdh is used as an endogenous control. For GFI1 knockdown, GFI1 (A), *** P = 0.001, TRIM37 (A), ns P = 0.831, GFI1 ( G ), * P = 0.024, and TRIM37 (G), ns P = 0.345. For PAX5 knockdown, PAX5 (A), *** P = 0.0005, TRIM37 (A), ns P = 0.187, PAX5 ( G ), *** P = 0.001, and TRIM37 ( G ), ns P = 0.874, unpaired t test. Data are mean±SD of biological replicates, n ≥3/group. ( G ) Immunoblot for biotin-labeled TRIM37 enhancer fragment incubated with nuclear extract from control or NRF1 knockdown HEK293T cells and unlabeled probe (competitor) as indicated. The bound and free DNA fragments are indicated with red arrows, and the shift in the band is indicated (red asterisk, *). The concentrations of nuclear extract (5 μg), Biotin-labeled probes (0.2 pmol), competitor (10 pmol), and poly [d(I-C)] (50 ng/μl) were indicated at the Top . The representative images from two different experiments (Replicate #1–2) are shown. ( H ) A looping assay measuring the promoter–enhancer interactions for in vitro synthesized DNA fragments (Chr17: 59,107,405-59,106,446) with risk or reference alleles or mutated NRF1 binding motifs in the promoter was used. For BSA, ns P = 0.947, NRF1, ** P = 0.003, NRF1 vs mutated NRF1, ** P = 0.009, and DNase, ns P = 0.965, unpaired t test. Data are mean±SD of biological replicates, n = 3/group. ( I ) Correlation plots of the FPKM read from RNA-seq analysis for each replicate are shown for control ( Left ) and 51-G ( Right ) cells. ( J ) Venn diagram showing the overlap between differentially expressed genes in control and 51-G cells. ( K ) qRT-PCR analysis monitoring TRIM37 in control and 51-G cells. Gapdh is used as an endogenous control. *** P = 0.0003, unpaired t test. Data are mean ± SD of biological replicates, n = 4/group. The boxed areas span the first to the third quartile. The whiskers represent the 15th and 85th percentiles. ( L ) The top ten pathways enriched in 51-G relative to control cells identified by GSEA are shown.

Article Snippet: Immunoblots were probed using antibodies against Gapdh (Thermofisher), TRIM37 (Cell Signaling), and RAS (Cell Signaling).

Techniques: Clone Assay, Western Blot, Labeling, Incubation, Binding Assay, Quantitative RT-PCR, Knockdown, Control, In Vitro, Synthesized, RNA Sequencing Assay

( A ) A schematic summary for amplicon sequencing of A- and G allele for rs57141087 inserted through CRISPR/Cas9 technology in KTB51 breast epithelial cells. ( B ) ChIP assay monitoring PolII recruitment on TRIM37 promoter in KTB51 cells with A or G allele for rs57141087. Actin is used as an endogenous control. For TRIM37, * P = 0.045, and Actin, ns P = 0.889, unpaired t test. Data are mean ± SD of biological replicates, n = 3/group. ( C ) Immunoblot for biotin-labeled TRIM37 enhancer fragment with risk (G allele) and reference (A allele) incubated with HEK293T nuclear extract and competitor (unlabeled probe) as indicated. The bound and free DNA fragments are indicated with red arrows. The concentrations of nuclear extract (5 μg), Biotin-labeled probes (0.2 pmol), competitor (10 pmol), and poly [d(I-C)] (50 ng/μl) were indicated at the top. The representative image from one experiment is shown (left), and the shift in the band is indicated (red asterisk, *) and quantitated (right). ** P = 0.004, unpaired t test. Data are mean±SD of biological replicates, n = 3/group. ( D ) The risk G allele of rs57141087 harboring NRF1 binding motif in the enhancer region of TRIM37 (Chr17: 59,107,267–59,107,262) is shown. ( E ) qRT-PCR monitoring NRF1 and TRIM37 levels in KTB51 with A or G allele for rs57141087. Gapdh is used as an endogenous control. For reference allele NRF1, *** P = 0.0001, and TRIM37, ns P = 0.230; For risk allele NRF1, *** P = 2.68 × 10 −8 , and TRIM37, *** P = 0.0001, unpaired t test. Data are mean ± SD of biological replicates, n ≥ 4/group. ( F ) ChIP assay monitoring NRF1 recruitment on TRIM37 locus in 51-G and 51-A cells. NRF1 non-binding site (NBS) is used as a negative control. The primers used for the qPCR analysis in the TRIM37 locus are indicated on Top . For Enhancer, *** P = 0.0007, Promoter, ns P = 0.544, and NBS, ns P = 0.149, unpaired t test. Data are mean±SD of biological replicates, n = 3/group. ( G ) Immunoblot for biotin-labeled TRIM37 enhancer fragment incubated with nuclear extract from control or NRF1 knockdown HEK293T cells and unlabeled probe (competitor) as indicated. The bound and free DNA fragments are indicated with red arrows. The concentrations of nuclear extract (5 μg), Biotin-labeled probes (0.2 pmol), competitor (10 pmol), and poly [d(I-C)] (50 ng/μl) were indicated at the top. The representative image from one experiment is shown (left), and the shift in the band is indicated (red asterisk, *) and quantitated (right). ** P = 0.006, unpaired t test. Data are mean±SD of biological replicates, n = 3/group. ( H ) 3C-qPCR analysis across the ~5500-bp TRIM37 locus harboring risk and reference allele of SNP rs57141087 in 51-G and 51-A cells, respectively. The schematic shows the TRIM37 gene structure with PCR primers used for ChIP-qPCR assay (top ) . For ( A ), ns P = 0.780, ( B ), ns P = 0.206, ( C ), ns P = 0.918, ( D ), ns P = 0.814, ( E ), ns P = 0.069; ( F ), ** P = 0.002, ( G ), ns P = 0.104, ( H ), ns P = 0.247, and ( I – K ), not applicable, paired t test. Data are mean ± SD of biological replicates, n = 3/group. ( I ) ChIP assay monitoring PolII recruitment on TRIM37 promoter in control and NRF1 knockdown KTB51 cells. Actin is used as an endogenous control. The primers used for the qPCR analysis in the TRIM37 locus are indicated on top. For Promoter, ** n = 0.005, and Actin, ns n = 0.551, unpaired t test. Data are mean ± SD of biological replicates, n = 4/group. ( J ) Volcano plot illustrates differential gene expression in KTB51 with A or G allele for rs57141087 ( n = 2 biological replicates). Differential gene expression between the groups was analyzed by DESeq2 Bioconductor. FDR < 0.05. Red are significantly upregulated genes ( n = 807), blue are significantly downregulated genes ( n = 837), and grey are genes not significantly changed ( n = 11,628). ( K ) Enrichment plots for cancer stem cells (left) and EMT (right) gene signatures identified through GSEA analysis of RNA-seq data. Nominal P values were computed using a t test. ( L ) Graphical representation of the TRIM37 promoter regulation by the risk allele of rs57141087 and its effect on breast cancer progression. .

Journal: EMBO Reports

Article Title: The TRIM37 variant rs57141087 contributes to triple-negative breast cancer outcomes in Black women

doi: 10.1038/s44319-024-00331-2

Figure Lengend Snippet: ( A ) A schematic summary for amplicon sequencing of A- and G allele for rs57141087 inserted through CRISPR/Cas9 technology in KTB51 breast epithelial cells. ( B ) ChIP assay monitoring PolII recruitment on TRIM37 promoter in KTB51 cells with A or G allele for rs57141087. Actin is used as an endogenous control. For TRIM37, * P = 0.045, and Actin, ns P = 0.889, unpaired t test. Data are mean ± SD of biological replicates, n = 3/group. ( C ) Immunoblot for biotin-labeled TRIM37 enhancer fragment with risk (G allele) and reference (A allele) incubated with HEK293T nuclear extract and competitor (unlabeled probe) as indicated. The bound and free DNA fragments are indicated with red arrows. The concentrations of nuclear extract (5 μg), Biotin-labeled probes (0.2 pmol), competitor (10 pmol), and poly [d(I-C)] (50 ng/μl) were indicated at the top. The representative image from one experiment is shown (left), and the shift in the band is indicated (red asterisk, *) and quantitated (right). ** P = 0.004, unpaired t test. Data are mean±SD of biological replicates, n = 3/group. ( D ) The risk G allele of rs57141087 harboring NRF1 binding motif in the enhancer region of TRIM37 (Chr17: 59,107,267–59,107,262) is shown. ( E ) qRT-PCR monitoring NRF1 and TRIM37 levels in KTB51 with A or G allele for rs57141087. Gapdh is used as an endogenous control. For reference allele NRF1, *** P = 0.0001, and TRIM37, ns P = 0.230; For risk allele NRF1, *** P = 2.68 × 10 −8 , and TRIM37, *** P = 0.0001, unpaired t test. Data are mean ± SD of biological replicates, n ≥ 4/group. ( F ) ChIP assay monitoring NRF1 recruitment on TRIM37 locus in 51-G and 51-A cells. NRF1 non-binding site (NBS) is used as a negative control. The primers used for the qPCR analysis in the TRIM37 locus are indicated on Top . For Enhancer, *** P = 0.0007, Promoter, ns P = 0.544, and NBS, ns P = 0.149, unpaired t test. Data are mean±SD of biological replicates, n = 3/group. ( G ) Immunoblot for biotin-labeled TRIM37 enhancer fragment incubated with nuclear extract from control or NRF1 knockdown HEK293T cells and unlabeled probe (competitor) as indicated. The bound and free DNA fragments are indicated with red arrows. The concentrations of nuclear extract (5 μg), Biotin-labeled probes (0.2 pmol), competitor (10 pmol), and poly [d(I-C)] (50 ng/μl) were indicated at the top. The representative image from one experiment is shown (left), and the shift in the band is indicated (red asterisk, *) and quantitated (right). ** P = 0.006, unpaired t test. Data are mean±SD of biological replicates, n = 3/group. ( H ) 3C-qPCR analysis across the ~5500-bp TRIM37 locus harboring risk and reference allele of SNP rs57141087 in 51-G and 51-A cells, respectively. The schematic shows the TRIM37 gene structure with PCR primers used for ChIP-qPCR assay (top ) . For ( A ), ns P = 0.780, ( B ), ns P = 0.206, ( C ), ns P = 0.918, ( D ), ns P = 0.814, ( E ), ns P = 0.069; ( F ), ** P = 0.002, ( G ), ns P = 0.104, ( H ), ns P = 0.247, and ( I – K ), not applicable, paired t test. Data are mean ± SD of biological replicates, n = 3/group. ( I ) ChIP assay monitoring PolII recruitment on TRIM37 promoter in control and NRF1 knockdown KTB51 cells. Actin is used as an endogenous control. The primers used for the qPCR analysis in the TRIM37 locus are indicated on top. For Promoter, ** n = 0.005, and Actin, ns n = 0.551, unpaired t test. Data are mean ± SD of biological replicates, n = 4/group. ( J ) Volcano plot illustrates differential gene expression in KTB51 with A or G allele for rs57141087 ( n = 2 biological replicates). Differential gene expression between the groups was analyzed by DESeq2 Bioconductor. FDR < 0.05. Red are significantly upregulated genes ( n = 807), blue are significantly downregulated genes ( n = 837), and grey are genes not significantly changed ( n = 11,628). ( K ) Enrichment plots for cancer stem cells (left) and EMT (right) gene signatures identified through GSEA analysis of RNA-seq data. Nominal P values were computed using a t test. ( L ) Graphical representation of the TRIM37 promoter regulation by the risk allele of rs57141087 and its effect on breast cancer progression. .

Article Snippet: Immunoblots were probed using antibodies against Gapdh (Thermofisher), TRIM37 (Cell Signaling), and RAS (Cell Signaling).

Techniques: Amplification, Sequencing, CRISPR, Control, Western Blot, Labeling, Incubation, Binding Assay, Quantitative RT-PCR, Negative Control, Knockdown, Expressing, RNA Sequencing Assay

( A ) Immunoblots in KTB51 and TRIM37 derivatives of RAS-transformed KTB51 (K51t) cells. Gapdh was the loading control. ( B ) Relative cell growth for KTB51, K51t, K51-TRIM37, and K51t-TRIM37 cells at indicated times. For 48 h, K51t, ns P = 0.234, K51-TRIM37, ns P = 0.413, and K51t-TRIM37, ns P = 0.108. For 120 h., K51t, *** P = 8.96 × 10 −5 , K51-TRIM37, ** P = 0.002, and K51t-TRIM37, *** P = 8.64 × 10 −5 . For 144 h., K51t, *** P = 0.0002, K51-TRIM37, *** P = 2.61 × 10 −5 , and K51t-TRIM37, *** P = 3.10 × 10 −5 , unpaired t test. Data are mean ± SD of biological replicates, n = 5/group. ( C ) Representative bright-field images after crystal violet staining showing the growth of KTB51, K51t, K51-TRIM37, and K51t-TRIM37 cells. The colonies were quantified (right). For K51t, * P = 0.012, K51-TRIM37, ** P = 0.005, and K51t-TRIM37, P = **0.009, unpaired t test. Data are mean±SD of biological replicates, n = 3/group. ( D ) The relative migratory abilities for KTB51, K51t, K51-TRIM37, and K51t-TRIM37 cells were quantitated after 4 h. For K51t, *** P = 3.3 × 10 −5 , K51-TRIM37, *** P = 6.44 × 10 −5 , and K51t-TRIM37, *** P = 5.26 × 10 −7 ; K51t vs. K51t-TRIM37, ns P = 0.138, K51-TRIM37 vs. K51t-TRIM37, ** P = 0.005, unpaired t test. Data are mean ± SD of biological replicates, n = 6/group. ( E ) Quantitation of solid mammospheres formed by KTB51, K51t, K51-TRIM37, and K51t-TRIM37 cells. For K51t, * P = 0.012, K51-TRIM37, *** P = 0.001, and K51t-TRIM37, ** P = 0.005; K51t vs. K51t-TRIM37, * P = 0.024, K51-TRIM37 vs. K51t-TRIM37, * P = 0.018, unpaired t test. Data are mean±SD of biological replicates, n = 6/group. ( F , G ) FACS analysis of CD24 and CD44 ( F ) and EpCAM ( G ) in KTB51, K51t, K51-TRIM37, and K51t-TRIM37 cells derived from mammospheres in ( E ). For CD44 high /CD24 low in K51t, * P = 0.026, K51-TRIM37, ** P = 0.003, and K51t-TRIM37, *** P = 1.31 × 10 −6 . For EpCAM + in K51t, * P = 0.011, K51-TRIM37, * P = 0.011, and K51t-TRIM37, ** P = 0.003, unpaired t test. Data are mean±SD of biological replicates, n = 3/group. ( H ) qRT-PCR analysis of EMT markers in KTB51, K51t, K51-TRIM37, and K51t-TRIM37 cells derived from mammospheres in ( E ). Gapdh is used as an endogenous control. For CDH1 in K51t, *** P = 8.08 × 10 −5 , K51-TRIM37, *** P = 1.70 × 10 −5 , and K51t-TRIM37, *** P = 0.0003; CDH2; K51t, *** P = 9.11 × 10 −5 , K51-TRIM37, *** P = 0.0004, and K51t-TRIM37, *** P = 0.0002, For SNAI1 in K51t, ** P = 0.003, K51-TRIM37, *** P = 2.8 × 10 −6 , and K51t-TRIM37, ** P = 0.005. For TWIST1 in K51t, * P = 0.012, K51-TRIM37, ** P = 0.0014, and K51t-TRIM37, * P = 0.022. For ZEB1 in K51t, * P = 0.019, K51-TRIM37, * P = 0.013, and K51t-TRIM37, * P = 0.020, unpaired t test. Data are mean ± SD of biological replicates, n = 3/group. ( I ) Quantitation of colonies formed in soft agar by KTB51, K51t, K51-TRIM37, and K51t-TRIM37 cells. For K51t, *** P = 0.0002, K51-TRIM37, ** P = 0.009, and K51t-TRIM37, *** P = 0.0005; K51t vs. K51t-TRIM37, ns P = 0.201, K51-TRIM37 vs. K51t-TRIM37, * P = 0.044, unpaired t test. Data are mean±SD of biological replicates, n = 3/group. ( J ) The xenograft tumor volume measurements of NSG mice injected with KTB51, K51t, K51-TRIM37, and K51t-TRIM37 cells. For 7 days, K51t, * P = 0.018, K51-TRIM37, * P = 0.033, and K51t-TRIM37, *** P = 6.28 × 10 −5 . For 10 days, K51t, * P = 0.011, K51-TRIM37, * P = 0.039, and K51t-TRIM37, ** P = 0.007. For 13 days, K51t, *** P = 1.71 × 10 −5 , K51-TRIM37, *** P = 0.0002, and K51t-TRIM37, *** P = 1.42 × 10 −5 . For 16 days, K51t, ** P = 0.002, K51-TRIM37, ** P = 0.005, and K51t-TRIM37, *** P = 1.85 × 10 −5 , K51t vs. K51t-TRIM37, ** P = 0.005, and K51-TRIM37 vs. K51t-TRIM37, ** P = 0.004, unpaired t test. Data are mean ± SD of biological replicates, n = 6/group. ( K , L ) Representative images of H&E ( K , ×20, scale bar, 200 μm) and Ki67 ( L , ×20, scale bar, 200 μm) for the tumors isolated in ( J ) are shown. ( M – O ) Representative immunohistochemical (IHC) images of ER ( M , ×20, scale bar, 200 μm), PR ( N , ×20, scale bar, 200 μm), and Her2 ( O , ×20, scale bar, 200 μm) for the tumors isolated in ( J ) are shown. The manual quantification of the IHC signal is shown for ER ( M , bottom), PR ( N , bottom), and Her2 ( O , bottom) ( n = ~1200–1500 cells per group). .

Journal: EMBO Reports

Article Title: The TRIM37 variant rs57141087 contributes to triple-negative breast cancer outcomes in Black women

doi: 10.1038/s44319-024-00331-2

Figure Lengend Snippet: ( A ) Immunoblots in KTB51 and TRIM37 derivatives of RAS-transformed KTB51 (K51t) cells. Gapdh was the loading control. ( B ) Relative cell growth for KTB51, K51t, K51-TRIM37, and K51t-TRIM37 cells at indicated times. For 48 h, K51t, ns P = 0.234, K51-TRIM37, ns P = 0.413, and K51t-TRIM37, ns P = 0.108. For 120 h., K51t, *** P = 8.96 × 10 −5 , K51-TRIM37, ** P = 0.002, and K51t-TRIM37, *** P = 8.64 × 10 −5 . For 144 h., K51t, *** P = 0.0002, K51-TRIM37, *** P = 2.61 × 10 −5 , and K51t-TRIM37, *** P = 3.10 × 10 −5 , unpaired t test. Data are mean ± SD of biological replicates, n = 5/group. ( C ) Representative bright-field images after crystal violet staining showing the growth of KTB51, K51t, K51-TRIM37, and K51t-TRIM37 cells. The colonies were quantified (right). For K51t, * P = 0.012, K51-TRIM37, ** P = 0.005, and K51t-TRIM37, P = **0.009, unpaired t test. Data are mean±SD of biological replicates, n = 3/group. ( D ) The relative migratory abilities for KTB51, K51t, K51-TRIM37, and K51t-TRIM37 cells were quantitated after 4 h. For K51t, *** P = 3.3 × 10 −5 , K51-TRIM37, *** P = 6.44 × 10 −5 , and K51t-TRIM37, *** P = 5.26 × 10 −7 ; K51t vs. K51t-TRIM37, ns P = 0.138, K51-TRIM37 vs. K51t-TRIM37, ** P = 0.005, unpaired t test. Data are mean ± SD of biological replicates, n = 6/group. ( E ) Quantitation of solid mammospheres formed by KTB51, K51t, K51-TRIM37, and K51t-TRIM37 cells. For K51t, * P = 0.012, K51-TRIM37, *** P = 0.001, and K51t-TRIM37, ** P = 0.005; K51t vs. K51t-TRIM37, * P = 0.024, K51-TRIM37 vs. K51t-TRIM37, * P = 0.018, unpaired t test. Data are mean±SD of biological replicates, n = 6/group. ( F , G ) FACS analysis of CD24 and CD44 ( F ) and EpCAM ( G ) in KTB51, K51t, K51-TRIM37, and K51t-TRIM37 cells derived from mammospheres in ( E ). For CD44 high /CD24 low in K51t, * P = 0.026, K51-TRIM37, ** P = 0.003, and K51t-TRIM37, *** P = 1.31 × 10 −6 . For EpCAM + in K51t, * P = 0.011, K51-TRIM37, * P = 0.011, and K51t-TRIM37, ** P = 0.003, unpaired t test. Data are mean±SD of biological replicates, n = 3/group. ( H ) qRT-PCR analysis of EMT markers in KTB51, K51t, K51-TRIM37, and K51t-TRIM37 cells derived from mammospheres in ( E ). Gapdh is used as an endogenous control. For CDH1 in K51t, *** P = 8.08 × 10 −5 , K51-TRIM37, *** P = 1.70 × 10 −5 , and K51t-TRIM37, *** P = 0.0003; CDH2; K51t, *** P = 9.11 × 10 −5 , K51-TRIM37, *** P = 0.0004, and K51t-TRIM37, *** P = 0.0002, For SNAI1 in K51t, ** P = 0.003, K51-TRIM37, *** P = 2.8 × 10 −6 , and K51t-TRIM37, ** P = 0.005. For TWIST1 in K51t, * P = 0.012, K51-TRIM37, ** P = 0.0014, and K51t-TRIM37, * P = 0.022. For ZEB1 in K51t, * P = 0.019, K51-TRIM37, * P = 0.013, and K51t-TRIM37, * P = 0.020, unpaired t test. Data are mean ± SD of biological replicates, n = 3/group. ( I ) Quantitation of colonies formed in soft agar by KTB51, K51t, K51-TRIM37, and K51t-TRIM37 cells. For K51t, *** P = 0.0002, K51-TRIM37, ** P = 0.009, and K51t-TRIM37, *** P = 0.0005; K51t vs. K51t-TRIM37, ns P = 0.201, K51-TRIM37 vs. K51t-TRIM37, * P = 0.044, unpaired t test. Data are mean±SD of biological replicates, n = 3/group. ( J ) The xenograft tumor volume measurements of NSG mice injected with KTB51, K51t, K51-TRIM37, and K51t-TRIM37 cells. For 7 days, K51t, * P = 0.018, K51-TRIM37, * P = 0.033, and K51t-TRIM37, *** P = 6.28 × 10 −5 . For 10 days, K51t, * P = 0.011, K51-TRIM37, * P = 0.039, and K51t-TRIM37, ** P = 0.007. For 13 days, K51t, *** P = 1.71 × 10 −5 , K51-TRIM37, *** P = 0.0002, and K51t-TRIM37, *** P = 1.42 × 10 −5 . For 16 days, K51t, ** P = 0.002, K51-TRIM37, ** P = 0.005, and K51t-TRIM37, *** P = 1.85 × 10 −5 , K51t vs. K51t-TRIM37, ** P = 0.005, and K51-TRIM37 vs. K51t-TRIM37, ** P = 0.004, unpaired t test. Data are mean ± SD of biological replicates, n = 6/group. ( K , L ) Representative images of H&E ( K , ×20, scale bar, 200 μm) and Ki67 ( L , ×20, scale bar, 200 μm) for the tumors isolated in ( J ) are shown. ( M – O ) Representative immunohistochemical (IHC) images of ER ( M , ×20, scale bar, 200 μm), PR ( N , ×20, scale bar, 200 μm), and Her2 ( O , ×20, scale bar, 200 μm) for the tumors isolated in ( J ) are shown. The manual quantification of the IHC signal is shown for ER ( M , bottom), PR ( N , bottom), and Her2 ( O , bottom) ( n = ~1200–1500 cells per group). .

Article Snippet: Immunoblots were probed using antibodies against Gapdh (Thermofisher), TRIM37 (Cell Signaling), and RAS (Cell Signaling).

Techniques: Western Blot, Transformation Assay, Control, Staining, Quantitation Assay, Derivative Assay, Quantitative RT-PCR, Injection, Isolation, Immunohistochemical staining

( A ) Representative phase contrast images of relative migratory abilities for KTB51, K51t, K51-TRIM37, and K51t-TRIM37 after 4 h. (10X, scale bar, 300 μm). ( B ) Representative images of solid mammospheres formed by KTB51, K51t, K51-TRIM37, and K51t-TRIM37 cells. (10X, scale bar, 300 μm). ( C , D ) Representative FACS plot showing gating strategy and distribution of stained population for CD24 and CD44 ( C ) and EpCAM ( D ) in KTB51, K51t, K51-TRIM37, and K51t-TRIM37 cells. ( E ) Immunoblots in KTB37 and TRIM37 derivatives of RAS-transformed KTB37(K37t) cells. Gapdh was the loading control. ( F ) Relative cell growth for KTB37 and TRIM37 derivatives of RAS-transformed KTB37 (K37t-TRIM37) cells at indicated times. For 48 h., K37t, ns P = 0.655, K37-TRIM37, ns P = 0.287, and K37t-TRIM37, ns P = 0.058. For 120 h., K37t, ** P = 0.006, K37-TRIM37, ** P = 0.003, and K37t-TRIM37, *** P = 0.0003. For 144 h. K37t, ** P = 0.004, K37-TRIM37, ** P = 0.009, and K37t-TRIM37, *** P = 0.0002, unpaired t test. Data are mean ± SD of biological replicates, n = 3/group. ( G ) Representative bright-field images after crystal violet staining show the growth of KTB37, K37t, K37-TRIM37, and K37t-TRIM37 cells. The colonies were quantified ( Right ). For K37t, *** P = 0.0005, K37-TRIM37, ** P = 0.003, and K37t-TRIM37, *** P = 1.86*10 -5 , unpaired t test. Data are mean ± SD of biological replicates, n = 3/group. ( H ) The relative migratory abilities for KTB37, K37t, K37-TRIM37, and K37t-TRIM37 cells were quantitated after 4 h. For K37t, *** P = 1.32*10 -13 , K37-TRIM37, *** P = 3.39*10 -14 , and K37t-TRIM37, *** P = 1.72*10 -16 ; K37t vs. K37t-TRIM37, *** P = 4.13*10 -5 , K37-TRIM37 vs. K37t-TRIM37, *** P = 1.28*10 -6 , unpaired t test. Data are mean±SD of biological replicates, n = 6/group. ( I , J ) Representative images (10X, scale bar, 300 μm) ( I ) and quantitation ( J ) of solid mammospheres formed by KTB37, K37t, K37-TRIM37, and K37t-TRIM37 cells. The colonies were quantified. For K37t, ** P = 0.003, K37-TRIM37, ** P = 0.004, and K37t-TRIM37, *** P = 2.15*10 -5 ; K37t vs. K37t-TRIM37, * P = 0.021, K37-TRIM37 vs. K37t-TRIM37, *** P = 0.0007, unpaired t test. Data are mean±SD of biological replicates, n = 3/group. ( K , L ) FACS analysis of CD24 and CD44 ( K ) and EpCAM ( L ) in KTB37, K37t, K37-TRIM37, and K37t-TRIM37 cells derived from mammospheres in ( I , J ). For CD44 high /CD24 low in K37t, * P = 0.036, K37-TRIM37, *** P = 0.0001, and K37t-TRIM37, *** P = 0.0008. For EpCAM + in K37t, *** P = 1.99*10 -5 , K37-TRIM37, * P = 0.028, and K37t-TRIM37, * P = 0.024, unpaired t test. Data are mean±SD of biological replicates, n = 3/group. ( M ) qRT-PCR analysis of EMT markers in KTB37, K37t, K37-TRIM37, and K37t-TRIM37 cells derived from mammospheres in ( I , J ). Gapdh is used as an endogenous control. For CDH1 in K37t, *** P = 2.03*10 -7 , K37-TRIM37, *** P = 1.01*10 -6 , and K37t-TRIM37, *** P = 1.58*10 -5 . For CDH2 in K37t, *** P = 5.52*10 -5 , K37-TRIM37, *** P = 0.0001, and K37t-TRIM37, *** P = 1.38*10 -5 . For SNAI1 in K37t, *** P = 0.0002, K37-TRIM37, *** P = 2.56*10 -5 , and K37t-TRIM37, *** P = 0.0006. For TWIST1 in K37t, *** P = 3.08*10 -5 , K37-TRIM37, *** P = 0.0003, and K37t-TRIM37, ** P = 0.002. For ZEB1 in K37t, *** P = 0.0001, K37-TRIM37, ns P = 0.051, and K37t-TRIM37, *** P = 0.0002, unpaired t test. Data are mean±SD of biological replicates, n = 3/group. ( N ) Representative bright-field images showing gross histology of xenografts harvested from NSG mice injected with, K51t, K51-TRIM37, and K51t-TRIM37 cells.

Journal: EMBO Reports

Article Title: The TRIM37 variant rs57141087 contributes to triple-negative breast cancer outcomes in Black women

doi: 10.1038/s44319-024-00331-2

Figure Lengend Snippet: ( A ) Representative phase contrast images of relative migratory abilities for KTB51, K51t, K51-TRIM37, and K51t-TRIM37 after 4 h. (10X, scale bar, 300 μm). ( B ) Representative images of solid mammospheres formed by KTB51, K51t, K51-TRIM37, and K51t-TRIM37 cells. (10X, scale bar, 300 μm). ( C , D ) Representative FACS plot showing gating strategy and distribution of stained population for CD24 and CD44 ( C ) and EpCAM ( D ) in KTB51, K51t, K51-TRIM37, and K51t-TRIM37 cells. ( E ) Immunoblots in KTB37 and TRIM37 derivatives of RAS-transformed KTB37(K37t) cells. Gapdh was the loading control. ( F ) Relative cell growth for KTB37 and TRIM37 derivatives of RAS-transformed KTB37 (K37t-TRIM37) cells at indicated times. For 48 h., K37t, ns P = 0.655, K37-TRIM37, ns P = 0.287, and K37t-TRIM37, ns P = 0.058. For 120 h., K37t, ** P = 0.006, K37-TRIM37, ** P = 0.003, and K37t-TRIM37, *** P = 0.0003. For 144 h. K37t, ** P = 0.004, K37-TRIM37, ** P = 0.009, and K37t-TRIM37, *** P = 0.0002, unpaired t test. Data are mean ± SD of biological replicates, n = 3/group. ( G ) Representative bright-field images after crystal violet staining show the growth of KTB37, K37t, K37-TRIM37, and K37t-TRIM37 cells. The colonies were quantified ( Right ). For K37t, *** P = 0.0005, K37-TRIM37, ** P = 0.003, and K37t-TRIM37, *** P = 1.86*10 -5 , unpaired t test. Data are mean ± SD of biological replicates, n = 3/group. ( H ) The relative migratory abilities for KTB37, K37t, K37-TRIM37, and K37t-TRIM37 cells were quantitated after 4 h. For K37t, *** P = 1.32*10 -13 , K37-TRIM37, *** P = 3.39*10 -14 , and K37t-TRIM37, *** P = 1.72*10 -16 ; K37t vs. K37t-TRIM37, *** P = 4.13*10 -5 , K37-TRIM37 vs. K37t-TRIM37, *** P = 1.28*10 -6 , unpaired t test. Data are mean±SD of biological replicates, n = 6/group. ( I , J ) Representative images (10X, scale bar, 300 μm) ( I ) and quantitation ( J ) of solid mammospheres formed by KTB37, K37t, K37-TRIM37, and K37t-TRIM37 cells. The colonies were quantified. For K37t, ** P = 0.003, K37-TRIM37, ** P = 0.004, and K37t-TRIM37, *** P = 2.15*10 -5 ; K37t vs. K37t-TRIM37, * P = 0.021, K37-TRIM37 vs. K37t-TRIM37, *** P = 0.0007, unpaired t test. Data are mean±SD of biological replicates, n = 3/group. ( K , L ) FACS analysis of CD24 and CD44 ( K ) and EpCAM ( L ) in KTB37, K37t, K37-TRIM37, and K37t-TRIM37 cells derived from mammospheres in ( I , J ). For CD44 high /CD24 low in K37t, * P = 0.036, K37-TRIM37, *** P = 0.0001, and K37t-TRIM37, *** P = 0.0008. For EpCAM + in K37t, *** P = 1.99*10 -5 , K37-TRIM37, * P = 0.028, and K37t-TRIM37, * P = 0.024, unpaired t test. Data are mean±SD of biological replicates, n = 3/group. ( M ) qRT-PCR analysis of EMT markers in KTB37, K37t, K37-TRIM37, and K37t-TRIM37 cells derived from mammospheres in ( I , J ). Gapdh is used as an endogenous control. For CDH1 in K37t, *** P = 2.03*10 -7 , K37-TRIM37, *** P = 1.01*10 -6 , and K37t-TRIM37, *** P = 1.58*10 -5 . For CDH2 in K37t, *** P = 5.52*10 -5 , K37-TRIM37, *** P = 0.0001, and K37t-TRIM37, *** P = 1.38*10 -5 . For SNAI1 in K37t, *** P = 0.0002, K37-TRIM37, *** P = 2.56*10 -5 , and K37t-TRIM37, *** P = 0.0006. For TWIST1 in K37t, *** P = 3.08*10 -5 , K37-TRIM37, *** P = 0.0003, and K37t-TRIM37, ** P = 0.002. For ZEB1 in K37t, *** P = 0.0001, K37-TRIM37, ns P = 0.051, and K37t-TRIM37, *** P = 0.0002, unpaired t test. Data are mean±SD of biological replicates, n = 3/group. ( N ) Representative bright-field images showing gross histology of xenografts harvested from NSG mice injected with, K51t, K51-TRIM37, and K51t-TRIM37 cells.

Article Snippet: Immunoblots were probed using antibodies against Gapdh (Thermofisher), TRIM37 (Cell Signaling), and RAS (Cell Signaling).

Techniques: Staining, Western Blot, Transformation Assay, Control, Quantitation Assay, Derivative Assay, Quantitative RT-PCR, Injection

Reagents and tools table

Journal: EMBO Reports

Article Title: The TRIM37 variant rs57141087 contributes to triple-negative breast cancer outcomes in Black women

doi: 10.1038/s44319-024-00331-2

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Immunoblots were probed using antibodies against Gapdh (Thermofisher), TRIM37 (Cell Signaling), and RAS (Cell Signaling).

Techniques: Recombinant, Plasmid Preparation, Purification, Sequencing, Mutagenesis, ChIP-chip, Clone Assay, shRNA, Software, Imaging

(A) A diagram depicting TRIM37 dysregulation in MUL and 17q23-amplified cancers. In MUL, TRIM37 loss-of-function mutations result in the aberrant formation of Centrobin assemblies, which act as ectopic MTOCs during mitosis. Conversely, in 17q23-amplified cancers, elevated expression of TRIM37 leads to excessive degradation of centrosomal CEP192. (B) Centre, domain architecture of TRIM37 (UniProt ID 094972) highlighting the common RBCC motif (RING, B-box, Coiled-coil domains) and unique C-terminal TRAF domain. Surrounding panels, localization pattern and effect of HA-tagged TRIM37 variants (domain-specific mutations and deletions) on centrosomal CEP192 levels in RPE-1 tet-on TRIM37 cells. MUL indicates Mulibrey nanism patient-derived mutations. Representative data; n = 3 biological replicates. Scale bars, 5 μm. (C) Quantification of Centrobin assembly occurrence in RPE-1 TRIM37 -/- cells expressing the indicated HA-tagged TRIM37 variants. n = 3 biological replicates, each with >100 cells. P values, one-way ANOVA with post hoc Dunnett’s multiple comparisons test to evaluate differences between the TRIM37 variants and wild-type (WT). Mean ± s.e.m. (D) Quantification of centrosomal CEP192 signal in RPE-1 tet-on TRIM37 cells from (B). n = 3 biological replicates, each with >100 cells. P values, unpaired two-tailed t -test. Mean ± s.e.m. (E) Left, AlphaFold-predicted monomer of TRIM37. The RING, B-box, Coiled-coil, and TRAF domains are shown, with mutated residues highlighted in red. Right, AlphaFold Multimer-predicted model of a TRIM37 dimer. For both models, the unstructured C-terminal tail of TRIM37 (residues 449–964) is not shown due to the lack of a high-confidence prediction.

Journal: bioRxiv

Article Title: Mesoscale regulation of MTOCs by the E3 ligase TRIM37

doi: 10.1101/2024.10.09.617407

Figure Lengend Snippet: (A) A diagram depicting TRIM37 dysregulation in MUL and 17q23-amplified cancers. In MUL, TRIM37 loss-of-function mutations result in the aberrant formation of Centrobin assemblies, which act as ectopic MTOCs during mitosis. Conversely, in 17q23-amplified cancers, elevated expression of TRIM37 leads to excessive degradation of centrosomal CEP192. (B) Centre, domain architecture of TRIM37 (UniProt ID 094972) highlighting the common RBCC motif (RING, B-box, Coiled-coil domains) and unique C-terminal TRAF domain. Surrounding panels, localization pattern and effect of HA-tagged TRIM37 variants (domain-specific mutations and deletions) on centrosomal CEP192 levels in RPE-1 tet-on TRIM37 cells. MUL indicates Mulibrey nanism patient-derived mutations. Representative data; n = 3 biological replicates. Scale bars, 5 μm. (C) Quantification of Centrobin assembly occurrence in RPE-1 TRIM37 -/- cells expressing the indicated HA-tagged TRIM37 variants. n = 3 biological replicates, each with >100 cells. P values, one-way ANOVA with post hoc Dunnett’s multiple comparisons test to evaluate differences between the TRIM37 variants and wild-type (WT). Mean ± s.e.m. (D) Quantification of centrosomal CEP192 signal in RPE-1 tet-on TRIM37 cells from (B). n = 3 biological replicates, each with >100 cells. P values, unpaired two-tailed t -test. Mean ± s.e.m. (E) Left, AlphaFold-predicted monomer of TRIM37. The RING, B-box, Coiled-coil, and TRAF domains are shown, with mutated residues highlighted in red. Right, AlphaFold Multimer-predicted model of a TRIM37 dimer. For both models, the unstructured C-terminal tail of TRIM37 (residues 449–964) is not shown due to the lack of a high-confidence prediction.

Article Snippet: Following electrophoresis, proteins were transferred onto nitrocellulose membranes using a Mini Trans-Blot Cell (BioRad) wet transfer system and subsequently probed with the following primary antibodies: TRIM37 (rabbit, Bethyl, A301-174A, 1:1000), HA (rat; Roche, ROAHAHA; 1:1000), β-actin (mouse, Santa Cruz Biotechnology, sc-4778, 1:1000), CEP192 (rabbit, raised against CEP192 residues 1–211, home-made , 1:1000), CNTROB (rabbit, Atlas Antibodies, HPA023319, 1:1000), SAS-6 (mouse, Santa Cruz Biotechnology, sc-81431, 1:1000), vinculin (mouse, Santa Cruz Biotechnology, sc-73614, 1:1000), GAPDH (mouse, Santa Cruz Biotechnology, sc-47724, 1:1000), TRIM37 (rabbit, Cell Signaling, #96167, 1:1000, see ), and mCherry (rabbit, Abcam, ab167453, 1:4000).

Techniques: Amplification, Expressing, Derivative Assay, Two Tailed Test

(A) Immunoblot showing TRIM37 protein levels in parental and CRISPR–Cas9 edited TRIM37 −/− RPE-1 cells. Ponceau-stained blot indicates loading. Representative data; n = 3 biological replicates. (B) Left, Tracking of Indels by Decomposition (TIDE) analysis histogram reveals a one base pair insertion (+1 bp) adjacent to the predicted cut site in the TRIM37 −/− RPE-1 cell line. Right, representative Sanger sequencing traces used for TIDE analysis, highlighting the +1 bp insertion. (C) Representative images of RPE-1 TRIM37 −/− cells and those expressing the indicated HA-tagged TRIM37 variants. Inset #1 denotes the centrosome, marked by CEP192, and inset #2 denotes the Centrobin assembly, identified by intense Centrobin staining that is non-centrosome localized. Representative data; n = 3 biological replicates. Scale bars, 5 μm. (D) Schematic representation of TRIM37 HA-tagged domain-specific deletion constructs compared to full-length (FL) protein. (E) Immunoblot showing expression levels of FL TRIM37 and the respective deletion mutants in RPE-1 tet-on TRIM37 cells. Ponceau-stained blot indicates loading. Representative data; n = 3 biological replicates.

Journal: bioRxiv

Article Title: Mesoscale regulation of MTOCs by the E3 ligase TRIM37

doi: 10.1101/2024.10.09.617407

Figure Lengend Snippet: (A) Immunoblot showing TRIM37 protein levels in parental and CRISPR–Cas9 edited TRIM37 −/− RPE-1 cells. Ponceau-stained blot indicates loading. Representative data; n = 3 biological replicates. (B) Left, Tracking of Indels by Decomposition (TIDE) analysis histogram reveals a one base pair insertion (+1 bp) adjacent to the predicted cut site in the TRIM37 −/− RPE-1 cell line. Right, representative Sanger sequencing traces used for TIDE analysis, highlighting the +1 bp insertion. (C) Representative images of RPE-1 TRIM37 −/− cells and those expressing the indicated HA-tagged TRIM37 variants. Inset #1 denotes the centrosome, marked by CEP192, and inset #2 denotes the Centrobin assembly, identified by intense Centrobin staining that is non-centrosome localized. Representative data; n = 3 biological replicates. Scale bars, 5 μm. (D) Schematic representation of TRIM37 HA-tagged domain-specific deletion constructs compared to full-length (FL) protein. (E) Immunoblot showing expression levels of FL TRIM37 and the respective deletion mutants in RPE-1 tet-on TRIM37 cells. Ponceau-stained blot indicates loading. Representative data; n = 3 biological replicates.

Article Snippet: Following electrophoresis, proteins were transferred onto nitrocellulose membranes using a Mini Trans-Blot Cell (BioRad) wet transfer system and subsequently probed with the following primary antibodies: TRIM37 (rabbit, Bethyl, A301-174A, 1:1000), HA (rat; Roche, ROAHAHA; 1:1000), β-actin (mouse, Santa Cruz Biotechnology, sc-4778, 1:1000), CEP192 (rabbit, raised against CEP192 residues 1–211, home-made , 1:1000), CNTROB (rabbit, Atlas Antibodies, HPA023319, 1:1000), SAS-6 (mouse, Santa Cruz Biotechnology, sc-81431, 1:1000), vinculin (mouse, Santa Cruz Biotechnology, sc-73614, 1:1000), GAPDH (mouse, Santa Cruz Biotechnology, sc-47724, 1:1000), TRIM37 (rabbit, Cell Signaling, #96167, 1:1000, see ), and mCherry (rabbit, Abcam, ab167453, 1:4000).

Techniques: Western Blot, CRISPR, Staining, Sequencing, Expressing, Construct

(A) Top, schematic of miniTurbo-TRIM37 construct used for BioID labelling experiments. Bottom, depiction of the approach to isolate TRAF domain-specific interactors of TRIM37. (B) Immunofluorescence images of biotin-labelled proteins in RPE-1 cells expressing the indicated mTurbo constructs. Streptavidin staining indicates biotinylated proteins, with centrosomes marked by CEP192 staining. Representative data; n = 2 biological replicates. Scale bars, 5 μm. (C) Thresholded mass spectrometry results displaying the top 34 proximity interactors (TRAF-domain specific) by spectral count. Interactors were identified using filters detailed in the Methods section, highlighting differential labelling by TRIM37 mutants after background subtraction. (D) Venn diagram illustrating the overlap between TRIM37 TRAF domain-specific proximity interactome (this study) and two published centrosome proximity interactomes. Accompanying list specifies hits common to the TRAF-domain interactome. (E) Gene ontology analysis of mass spectrometry data from BioID experiments.

Journal: bioRxiv

Article Title: Mesoscale regulation of MTOCs by the E3 ligase TRIM37

doi: 10.1101/2024.10.09.617407

Figure Lengend Snippet: (A) Top, schematic of miniTurbo-TRIM37 construct used for BioID labelling experiments. Bottom, depiction of the approach to isolate TRAF domain-specific interactors of TRIM37. (B) Immunofluorescence images of biotin-labelled proteins in RPE-1 cells expressing the indicated mTurbo constructs. Streptavidin staining indicates biotinylated proteins, with centrosomes marked by CEP192 staining. Representative data; n = 2 biological replicates. Scale bars, 5 μm. (C) Thresholded mass spectrometry results displaying the top 34 proximity interactors (TRAF-domain specific) by spectral count. Interactors were identified using filters detailed in the Methods section, highlighting differential labelling by TRIM37 mutants after background subtraction. (D) Venn diagram illustrating the overlap between TRIM37 TRAF domain-specific proximity interactome (this study) and two published centrosome proximity interactomes. Accompanying list specifies hits common to the TRAF-domain interactome. (E) Gene ontology analysis of mass spectrometry data from BioID experiments.

Article Snippet: Following electrophoresis, proteins were transferred onto nitrocellulose membranes using a Mini Trans-Blot Cell (BioRad) wet transfer system and subsequently probed with the following primary antibodies: TRIM37 (rabbit, Bethyl, A301-174A, 1:1000), HA (rat; Roche, ROAHAHA; 1:1000), β-actin (mouse, Santa Cruz Biotechnology, sc-4778, 1:1000), CEP192 (rabbit, raised against CEP192 residues 1–211, home-made , 1:1000), CNTROB (rabbit, Atlas Antibodies, HPA023319, 1:1000), SAS-6 (mouse, Santa Cruz Biotechnology, sc-81431, 1:1000), vinculin (mouse, Santa Cruz Biotechnology, sc-73614, 1:1000), GAPDH (mouse, Santa Cruz Biotechnology, sc-47724, 1:1000), TRIM37 (rabbit, Cell Signaling, #96167, 1:1000, see ), and mCherry (rabbit, Abcam, ab167453, 1:4000).

Techniques: Construct, Immunofluorescence, Expressing, Staining, Mass Spectrometry

(A) Schematic overview of the domain swap strategy, which replaces the TRIM5 SPRY domain with the TRIM37 TRAF domain to generate a chimeric TRIM5-TRAF protein. (B) Immunoblot showing total protein expression levels of indicated HA-tagged TRIM5 constructs in RPE-1 tet-on TRIM5 cells. Actin, loading control. Representative data; n = 3 biological replicates. (C) Representative images of the localization and effect of indicated HA-tagged TRIM5 constructs on centrosomal CEP192 levels in RPE-1 tet-on TRIM5 cells. Representative data; n = 3 biological replicates. Scale bars, 5 μm. (D) Quantification of centrosomal CEP192 signal upon doxycycline-induced expression of indicated constructs in RPE-1 tet-on TRIM5 cells from (C), with TRIM37 included as a benchmark. n = 3 biological replicates, each with >100 cells. P values, one-way ANOVA with post hoc Tukey’s multiple comparisons test. Mean ± s.e.m. (E) Representative images of RPE-1 TRIM37 −/− cells expressing the indicated HA-tagged TRIM5 constructs. Inset #1 denotes the centrosome, marked by CEP192, and inset #2 denotes the Centrobin assembly, identified by intense Centrobin staining that is non-centrosome localized. Representative data; n = 3 biological replicates. Scale bars, 5 μm. (F) Quantification of Centrobin assembly occurrence in RPE-1 TRIM37 -/- cells expressing the indicated HA-tagged TRIM5 constructs from (E). n = 3 biological replicates, each with >100 cells.

Journal: bioRxiv

Article Title: Mesoscale regulation of MTOCs by the E3 ligase TRIM37

doi: 10.1101/2024.10.09.617407

Figure Lengend Snippet: (A) Schematic overview of the domain swap strategy, which replaces the TRIM5 SPRY domain with the TRIM37 TRAF domain to generate a chimeric TRIM5-TRAF protein. (B) Immunoblot showing total protein expression levels of indicated HA-tagged TRIM5 constructs in RPE-1 tet-on TRIM5 cells. Actin, loading control. Representative data; n = 3 biological replicates. (C) Representative images of the localization and effect of indicated HA-tagged TRIM5 constructs on centrosomal CEP192 levels in RPE-1 tet-on TRIM5 cells. Representative data; n = 3 biological replicates. Scale bars, 5 μm. (D) Quantification of centrosomal CEP192 signal upon doxycycline-induced expression of indicated constructs in RPE-1 tet-on TRIM5 cells from (C), with TRIM37 included as a benchmark. n = 3 biological replicates, each with >100 cells. P values, one-way ANOVA with post hoc Tukey’s multiple comparisons test. Mean ± s.e.m. (E) Representative images of RPE-1 TRIM37 −/− cells expressing the indicated HA-tagged TRIM5 constructs. Inset #1 denotes the centrosome, marked by CEP192, and inset #2 denotes the Centrobin assembly, identified by intense Centrobin staining that is non-centrosome localized. Representative data; n = 3 biological replicates. Scale bars, 5 μm. (F) Quantification of Centrobin assembly occurrence in RPE-1 TRIM37 -/- cells expressing the indicated HA-tagged TRIM5 constructs from (E). n = 3 biological replicates, each with >100 cells.

Article Snippet: Following electrophoresis, proteins were transferred onto nitrocellulose membranes using a Mini Trans-Blot Cell (BioRad) wet transfer system and subsequently probed with the following primary antibodies: TRIM37 (rabbit, Bethyl, A301-174A, 1:1000), HA (rat; Roche, ROAHAHA; 1:1000), β-actin (mouse, Santa Cruz Biotechnology, sc-4778, 1:1000), CEP192 (rabbit, raised against CEP192 residues 1–211, home-made , 1:1000), CNTROB (rabbit, Atlas Antibodies, HPA023319, 1:1000), SAS-6 (mouse, Santa Cruz Biotechnology, sc-81431, 1:1000), vinculin (mouse, Santa Cruz Biotechnology, sc-73614, 1:1000), GAPDH (mouse, Santa Cruz Biotechnology, sc-47724, 1:1000), TRIM37 (rabbit, Cell Signaling, #96167, 1:1000, see ), and mCherry (rabbit, Abcam, ab167453, 1:4000).

Techniques: Western Blot, Expressing, Construct, Control, Staining

(A) Immunoblot showing expression levels of wild-type (WT) TRIM37 and indicated mutants in RPE-1 tet-on TRIM37 cells. Higher molecular weight (HMW) TRIM37 species are prominently formed in the C18R mutant and indicated with an arrow. β-Actin, loading control. Representative data; n = 3 biological replicates. (B) Same as in (A) but with MG132 (10 μM) treatment to achieve proteasomal inhibition and stabilization of WT TRIM37. β-Actin, loading control. Representative data; n = 3 biological replicates. (C) Top, immunoblot showing detection of various higher molecular weight (HMW) species of TRIM37 upon treatment with increasing concentrations of DSS crosslinker. Vinculin is used as a loading and oligomerization control. Dotted lines indicate separate cropped sections of the same immunoblot. Representative data; n = 3 biological replicates. Bottom, Densitometric analysis of immunoblot with a graph depicting normalized HMW TRIM37 intensity upon increasing DSS concentrations relative to DMSO control (−DSS). Mean ± s.e.m. (D) Representative Sanger sequencing traces of the TRIM37 locus in parental and CRISPR–Cas9 edited RPE-1 TRIM37 C18R cells, highlighting the mutation (TGT>CGT) responsible for the biallelic C18R residue substitution, denoted by an asterisk. (E) Left, immunoblot showing endogenous TRIM37 protein levels across the indicated cellular fractions from RPE-1 TRIM37 C18R cells. Validation markers include CEP192, Centrobin, and SAS6 for centrosomal proteins, and Lamin A/C for the nuclear fraction. Ponceau-stained blot indicates loading. Representative data; n = 3 biological replicates. WCE, whole-cell extract; exp, exposure. Right, Densitometric analysis of immunoblot in with a graph depicting endogenous TRIM37 enrichment in indicated fractions relative to WCE. P values, one-way ANOVA with post hoc Dunnett’s multiple comparisons test to evaluate enrichment of TRIM37 in each cellular fraction relative to WCE. Mean ± s.e.m. (F) Left, immunoblot showing detection of various higher molecular weight (HMW) species of endogenous TRIM37 upon treatment of RPE-1 TRIM37 C18R cells with increasing concentrations of DSG crosslinker. Vinculin is used as a loading and oligomerization control. Representative data; n = 3 biological replicates. Right, Densitometric analysis of immunoblot with a graph depicting normalized HMW TRIM37 intensity upon increasing DSG concentrations relative to DMSO control (−DSG). Mean ± s.e.m.

Journal: bioRxiv

Article Title: Mesoscale regulation of MTOCs by the E3 ligase TRIM37

doi: 10.1101/2024.10.09.617407

Figure Lengend Snippet: (A) Immunoblot showing expression levels of wild-type (WT) TRIM37 and indicated mutants in RPE-1 tet-on TRIM37 cells. Higher molecular weight (HMW) TRIM37 species are prominently formed in the C18R mutant and indicated with an arrow. β-Actin, loading control. Representative data; n = 3 biological replicates. (B) Same as in (A) but with MG132 (10 μM) treatment to achieve proteasomal inhibition and stabilization of WT TRIM37. β-Actin, loading control. Representative data; n = 3 biological replicates. (C) Top, immunoblot showing detection of various higher molecular weight (HMW) species of TRIM37 upon treatment with increasing concentrations of DSS crosslinker. Vinculin is used as a loading and oligomerization control. Dotted lines indicate separate cropped sections of the same immunoblot. Representative data; n = 3 biological replicates. Bottom, Densitometric analysis of immunoblot with a graph depicting normalized HMW TRIM37 intensity upon increasing DSS concentrations relative to DMSO control (−DSS). Mean ± s.e.m. (D) Representative Sanger sequencing traces of the TRIM37 locus in parental and CRISPR–Cas9 edited RPE-1 TRIM37 C18R cells, highlighting the mutation (TGT>CGT) responsible for the biallelic C18R residue substitution, denoted by an asterisk. (E) Left, immunoblot showing endogenous TRIM37 protein levels across the indicated cellular fractions from RPE-1 TRIM37 C18R cells. Validation markers include CEP192, Centrobin, and SAS6 for centrosomal proteins, and Lamin A/C for the nuclear fraction. Ponceau-stained blot indicates loading. Representative data; n = 3 biological replicates. WCE, whole-cell extract; exp, exposure. Right, Densitometric analysis of immunoblot in with a graph depicting endogenous TRIM37 enrichment in indicated fractions relative to WCE. P values, one-way ANOVA with post hoc Dunnett’s multiple comparisons test to evaluate enrichment of TRIM37 in each cellular fraction relative to WCE. Mean ± s.e.m. (F) Left, immunoblot showing detection of various higher molecular weight (HMW) species of endogenous TRIM37 upon treatment of RPE-1 TRIM37 C18R cells with increasing concentrations of DSG crosslinker. Vinculin is used as a loading and oligomerization control. Representative data; n = 3 biological replicates. Right, Densitometric analysis of immunoblot with a graph depicting normalized HMW TRIM37 intensity upon increasing DSG concentrations relative to DMSO control (−DSG). Mean ± s.e.m.

Article Snippet: Following electrophoresis, proteins were transferred onto nitrocellulose membranes using a Mini Trans-Blot Cell (BioRad) wet transfer system and subsequently probed with the following primary antibodies: TRIM37 (rabbit, Bethyl, A301-174A, 1:1000), HA (rat; Roche, ROAHAHA; 1:1000), β-actin (mouse, Santa Cruz Biotechnology, sc-4778, 1:1000), CEP192 (rabbit, raised against CEP192 residues 1–211, home-made , 1:1000), CNTROB (rabbit, Atlas Antibodies, HPA023319, 1:1000), SAS-6 (mouse, Santa Cruz Biotechnology, sc-81431, 1:1000), vinculin (mouse, Santa Cruz Biotechnology, sc-73614, 1:1000), GAPDH (mouse, Santa Cruz Biotechnology, sc-47724, 1:1000), TRIM37 (rabbit, Cell Signaling, #96167, 1:1000, see ), and mCherry (rabbit, Abcam, ab167453, 1:4000).

Techniques: Western Blot, Expressing, Molecular Weight, Mutagenesis, Control, Inhibition, Sequencing, CRISPR, Residue, Staining

(A) Schematic of the TRIM37 protein, highlighting epitopes recognized by two distinct commercial antibodies. (B-D) The commercial TRIM37 antibody (Bethyl, A301-173A) was utilized for the following experiments. (B) Immunoblot showing endogenous TRIM37 protein levels across a panel of cell lines with the indicated TRIM37 status. Ponceau-stained blot indicates loading. Representative data; n = 3 biological replicates. KI, knock-in; KO, knock-out; exp, exposure. (C) Representative images showing the immunostaining pattern of endogenous TRIM37 in the cell line panel. Arrows indicate the location of centrosomes. Representative data; n = 3 biological replicates. Scale bars, 5 μm. (D) Quantification of endogenous TRIM37 signal at the centrosomes of the cell line panel. n = 3 biological replicates, each with >100 cells. P values, one-way ANOVA with post hoc Tukey’s multiple comparisons test. Mean ± s.e.m. (E-G) Same as in (B-D), but with a second commercial antibody (Cell Signaling Technology, D7U2L).

Journal: bioRxiv

Article Title: Mesoscale regulation of MTOCs by the E3 ligase TRIM37

doi: 10.1101/2024.10.09.617407

Figure Lengend Snippet: (A) Schematic of the TRIM37 protein, highlighting epitopes recognized by two distinct commercial antibodies. (B-D) The commercial TRIM37 antibody (Bethyl, A301-173A) was utilized for the following experiments. (B) Immunoblot showing endogenous TRIM37 protein levels across a panel of cell lines with the indicated TRIM37 status. Ponceau-stained blot indicates loading. Representative data; n = 3 biological replicates. KI, knock-in; KO, knock-out; exp, exposure. (C) Representative images showing the immunostaining pattern of endogenous TRIM37 in the cell line panel. Arrows indicate the location of centrosomes. Representative data; n = 3 biological replicates. Scale bars, 5 μm. (D) Quantification of endogenous TRIM37 signal at the centrosomes of the cell line panel. n = 3 biological replicates, each with >100 cells. P values, one-way ANOVA with post hoc Tukey’s multiple comparisons test. Mean ± s.e.m. (E-G) Same as in (B-D), but with a second commercial antibody (Cell Signaling Technology, D7U2L).

Article Snippet: Following electrophoresis, proteins were transferred onto nitrocellulose membranes using a Mini Trans-Blot Cell (BioRad) wet transfer system and subsequently probed with the following primary antibodies: TRIM37 (rabbit, Bethyl, A301-174A, 1:1000), HA (rat; Roche, ROAHAHA; 1:1000), β-actin (mouse, Santa Cruz Biotechnology, sc-4778, 1:1000), CEP192 (rabbit, raised against CEP192 residues 1–211, home-made , 1:1000), CNTROB (rabbit, Atlas Antibodies, HPA023319, 1:1000), SAS-6 (mouse, Santa Cruz Biotechnology, sc-81431, 1:1000), vinculin (mouse, Santa Cruz Biotechnology, sc-73614, 1:1000), GAPDH (mouse, Santa Cruz Biotechnology, sc-47724, 1:1000), TRIM37 (rabbit, Cell Signaling, #96167, 1:1000, see ), and mCherry (rabbit, Abcam, ab167453, 1:4000).

Techniques: Western Blot, Staining, Knock-In, Knock-Out, Immunostaining

(A) Left, diagram illustrating the B-box trimerization interface of TRIM5 dimers on the HIV capsid. Trimers are stabilized by W117 residues within the hydrophobic core, as shown in the magnified top-down view of the TRIM5 B-box crystal structure (PDB 5VA4). Right, analogous diagram representing a putative oligomer formed by TRIM37 dimers at the centrosome, where B-box domain trimerization is hypothesized to be stabilized by W120 residues, the synonymous counterpart to TRIM5’s W117. A magnified top-down view shows the putative TRIM37 B-box trimer modelled by fitting AlphaFold-predicted TRIM37 monomers onto the TRIM5 crystal structure. (B) Comparative alignment of the B-box domains from human TRIM37 and human and rhesus macaque ( Macaca mulatta ) TRIM5. Residue Cys109, mutated in MUL disease, is pivotal for zinc (Zn) coordination. The highlighted region in grey denotes the sequence alignment where W115/W117 residues in TRIM5 correspond to the W120 residue in TRIM37, signifying a conserved structural motif critical for higher-order assembly. (C) Immunoblot showing total protein expression levels of TRIM37 variants in RPE-1 tet-on TRIM37 cells from (D). Vinculin, loading control. Representative data; n = 3 biological replicates. (D) Representative images of RPE-1 tet-on TRIM37 cells expressing the RING domain mutant TRIM37(C18R) or RING-B-box double mutants (C18R-C109S and C18R-W120E). Cells were treated with DMSO (control) or nocodazole (3.3 μM) 30 min before doxycycline induction to depolymerize microtubules. n = 3 biological replicates. Scale bars, 5 μm. (E) Quantification of centrosomal TRIM37 signal in DMSO-treated RPE-1 tet-on TRIM37 cells expressing the indicated TRIM37 variants from (D). n = 3 biological replicates, each with >100 cells. P values, one-way ANOVA with post hoc Dunnett’s multiple comparisons test to evaluate differences between each of the TRIM37 RING-B-box double mutants (C18R-C109S and C18R-W120E) and the RING mutant (C18R). Mean ± s.e.m. (F) Left, immunoblot showing detection of higher molecular weight (HMW) species of indicated TRIM37 variants upon in vivo DSG crosslinking. Vinculin is used as a loading and oligomerization control. Representative data; n = 3 biological replicates. Right, Densitometric analysis of immunoblot with graph depicting normalized HMW TRIM37 intensity with DSG crosslinker (+) relative to DMSO control (−DSG). Mean ± s.e.m. (G) Evaluation of cytoplasmic TRIM37 puncta prevalence in nocodazole (Noc)-treated RPE-1 tet-on TRIM37 cells expressing the indicated TRIM37 variants from (D). n = 3 biological replicates, each with >100 cells. P values, one-way ANOVA with post hoc Dunnett’s multiple comparisons test to evaluate differences between each of the TRIM37 RING-B-box double mutants (C18R-C109S and C18R-W120E) and the RING mutant (C18R). Mean ± s.e.m.

Journal: bioRxiv

Article Title: Mesoscale regulation of MTOCs by the E3 ligase TRIM37

doi: 10.1101/2024.10.09.617407

Figure Lengend Snippet: (A) Left, diagram illustrating the B-box trimerization interface of TRIM5 dimers on the HIV capsid. Trimers are stabilized by W117 residues within the hydrophobic core, as shown in the magnified top-down view of the TRIM5 B-box crystal structure (PDB 5VA4). Right, analogous diagram representing a putative oligomer formed by TRIM37 dimers at the centrosome, where B-box domain trimerization is hypothesized to be stabilized by W120 residues, the synonymous counterpart to TRIM5’s W117. A magnified top-down view shows the putative TRIM37 B-box trimer modelled by fitting AlphaFold-predicted TRIM37 monomers onto the TRIM5 crystal structure. (B) Comparative alignment of the B-box domains from human TRIM37 and human and rhesus macaque ( Macaca mulatta ) TRIM5. Residue Cys109, mutated in MUL disease, is pivotal for zinc (Zn) coordination. The highlighted region in grey denotes the sequence alignment where W115/W117 residues in TRIM5 correspond to the W120 residue in TRIM37, signifying a conserved structural motif critical for higher-order assembly. (C) Immunoblot showing total protein expression levels of TRIM37 variants in RPE-1 tet-on TRIM37 cells from (D). Vinculin, loading control. Representative data; n = 3 biological replicates. (D) Representative images of RPE-1 tet-on TRIM37 cells expressing the RING domain mutant TRIM37(C18R) or RING-B-box double mutants (C18R-C109S and C18R-W120E). Cells were treated with DMSO (control) or nocodazole (3.3 μM) 30 min before doxycycline induction to depolymerize microtubules. n = 3 biological replicates. Scale bars, 5 μm. (E) Quantification of centrosomal TRIM37 signal in DMSO-treated RPE-1 tet-on TRIM37 cells expressing the indicated TRIM37 variants from (D). n = 3 biological replicates, each with >100 cells. P values, one-way ANOVA with post hoc Dunnett’s multiple comparisons test to evaluate differences between each of the TRIM37 RING-B-box double mutants (C18R-C109S and C18R-W120E) and the RING mutant (C18R). Mean ± s.e.m. (F) Left, immunoblot showing detection of higher molecular weight (HMW) species of indicated TRIM37 variants upon in vivo DSG crosslinking. Vinculin is used as a loading and oligomerization control. Representative data; n = 3 biological replicates. Right, Densitometric analysis of immunoblot with graph depicting normalized HMW TRIM37 intensity with DSG crosslinker (+) relative to DMSO control (−DSG). Mean ± s.e.m. (G) Evaluation of cytoplasmic TRIM37 puncta prevalence in nocodazole (Noc)-treated RPE-1 tet-on TRIM37 cells expressing the indicated TRIM37 variants from (D). n = 3 biological replicates, each with >100 cells. P values, one-way ANOVA with post hoc Dunnett’s multiple comparisons test to evaluate differences between each of the TRIM37 RING-B-box double mutants (C18R-C109S and C18R-W120E) and the RING mutant (C18R). Mean ± s.e.m.

Article Snippet: Following electrophoresis, proteins were transferred onto nitrocellulose membranes using a Mini Trans-Blot Cell (BioRad) wet transfer system and subsequently probed with the following primary antibodies: TRIM37 (rabbit, Bethyl, A301-174A, 1:1000), HA (rat; Roche, ROAHAHA; 1:1000), β-actin (mouse, Santa Cruz Biotechnology, sc-4778, 1:1000), CEP192 (rabbit, raised against CEP192 residues 1–211, home-made , 1:1000), CNTROB (rabbit, Atlas Antibodies, HPA023319, 1:1000), SAS-6 (mouse, Santa Cruz Biotechnology, sc-81431, 1:1000), vinculin (mouse, Santa Cruz Biotechnology, sc-73614, 1:1000), GAPDH (mouse, Santa Cruz Biotechnology, sc-47724, 1:1000), TRIM37 (rabbit, Cell Signaling, #96167, 1:1000, see ), and mCherry (rabbit, Abcam, ab167453, 1:4000).

Techniques: Residue, Sequencing, Western Blot, Expressing, Control, Mutagenesis, Molecular Weight, In Vivo

(A) Schematic of the miniTRIM37 (RBCC-TRAF) construct compared to full-length TRIM37. (B) Representative images of the localization and effect of indicated HA-tagged TRIM37 constructs on centrosomal CEP192 levels in RPE-1 tet-on TRIM37 cells. Arrows indicate the location of centrosomes. Representative data; n = 3 biological replicates. Scale bars, 5 μm. (C) Quantification of centrosomal CEP192 signal upon doxycycline-induced expression of indicated HA-tagged TRIM37 constructs in RPE-1 tet-on TRIM37 cells from (B). n = 3 biological replicates, each with >100 cells. P values, one-way ANOVA with post hoc Tukey’s multiple comparisons test. Mean ± s.e.m. (D) Immunoblot showing total protein levels of indicated HA-tagged TRIM37 constructs in RPE-1 tet-on TRIM37 cells from (B-C). GAPDH, loading control. Representative data; n = 3 biological replicates. (E) Immunoblot showing detection of various higher molecular weight (HMW) species of miniTRIM37 upon treatment with increasing concentrations of DSG crosslinker. Vinculin is used as a loading and oligomerization control. Representative data; n = 3 biological replicates.

Journal: bioRxiv

Article Title: Mesoscale regulation of MTOCs by the E3 ligase TRIM37

doi: 10.1101/2024.10.09.617407

Figure Lengend Snippet: (A) Schematic of the miniTRIM37 (RBCC-TRAF) construct compared to full-length TRIM37. (B) Representative images of the localization and effect of indicated HA-tagged TRIM37 constructs on centrosomal CEP192 levels in RPE-1 tet-on TRIM37 cells. Arrows indicate the location of centrosomes. Representative data; n = 3 biological replicates. Scale bars, 5 μm. (C) Quantification of centrosomal CEP192 signal upon doxycycline-induced expression of indicated HA-tagged TRIM37 constructs in RPE-1 tet-on TRIM37 cells from (B). n = 3 biological replicates, each with >100 cells. P values, one-way ANOVA with post hoc Tukey’s multiple comparisons test. Mean ± s.e.m. (D) Immunoblot showing total protein levels of indicated HA-tagged TRIM37 constructs in RPE-1 tet-on TRIM37 cells from (B-C). GAPDH, loading control. Representative data; n = 3 biological replicates. (E) Immunoblot showing detection of various higher molecular weight (HMW) species of miniTRIM37 upon treatment with increasing concentrations of DSG crosslinker. Vinculin is used as a loading and oligomerization control. Representative data; n = 3 biological replicates.

Article Snippet: Following electrophoresis, proteins were transferred onto nitrocellulose membranes using a Mini Trans-Blot Cell (BioRad) wet transfer system and subsequently probed with the following primary antibodies: TRIM37 (rabbit, Bethyl, A301-174A, 1:1000), HA (rat; Roche, ROAHAHA; 1:1000), β-actin (mouse, Santa Cruz Biotechnology, sc-4778, 1:1000), CEP192 (rabbit, raised against CEP192 residues 1–211, home-made , 1:1000), CNTROB (rabbit, Atlas Antibodies, HPA023319, 1:1000), SAS-6 (mouse, Santa Cruz Biotechnology, sc-81431, 1:1000), vinculin (mouse, Santa Cruz Biotechnology, sc-73614, 1:1000), GAPDH (mouse, Santa Cruz Biotechnology, sc-47724, 1:1000), TRIM37 (rabbit, Cell Signaling, #96167, 1:1000, see ), and mCherry (rabbit, Abcam, ab167453, 1:4000).

Techniques: Construct, Expressing, Western Blot, Control, Molecular Weight

(A) Immunoblot showing TRIM37 protein levels in TP53 −/− MCF-7 cells. TRIM37 wild-type (WT), TRIM37 knockdown (KD) via shRNA, and cells harboring the C109S mutation in approximately half of the TRIM37 alleles present ( TRIM37 C109S ) were used. Vinculin, loading control. Representative data; n = 3 biological replicates. (B) Left, Representative data of a 10-d clonogenic survival of indicated MCF-7 cell lines from (A) treated with DMSO (control) or PLK4 inhibitor (PLK4i) (125 nM). Right, Quantification of relative growth in the presence PLK4i relative to DMSO. P values, one-way ANOVA with post hoc Dunnett’s multiple comparisons test to evaluate differences between each experimental condition (KD and C109S) and WT. Mean ± s.e.m (C) Quantification of mitotic CEP192 foci in PLK4i-treated TP53 −/− MCF-7 cells that lack centrosomes. n = 3, biological replicates, each comprising >30 cells. P values, one-way ANOVA with post hoc Dunnett’s multiple comparisons test to evaluate differences between each experimental condition (KD and C109S) and WT. Mean ± s.e.m (D) Representative images for (C). Scale bars, 5 μm. (E) Representative Sanger sequencing traces for the TRIM37 locus in parental TP53 −/− MCF-7 cells subjected to TRIM37 knockdown (KD) via shRNA, and CRISPR–Cas9 edited TRIM37 C109S KI cells. The mutation (TGT>TCT) leading to the C109S residue substitution is denoted by an asterisk. Silent mutations introduced to prevent re-editing are highlighted.

Journal: bioRxiv

Article Title: Mesoscale regulation of MTOCs by the E3 ligase TRIM37

doi: 10.1101/2024.10.09.617407

Figure Lengend Snippet: (A) Immunoblot showing TRIM37 protein levels in TP53 −/− MCF-7 cells. TRIM37 wild-type (WT), TRIM37 knockdown (KD) via shRNA, and cells harboring the C109S mutation in approximately half of the TRIM37 alleles present ( TRIM37 C109S ) were used. Vinculin, loading control. Representative data; n = 3 biological replicates. (B) Left, Representative data of a 10-d clonogenic survival of indicated MCF-7 cell lines from (A) treated with DMSO (control) or PLK4 inhibitor (PLK4i) (125 nM). Right, Quantification of relative growth in the presence PLK4i relative to DMSO. P values, one-way ANOVA with post hoc Dunnett’s multiple comparisons test to evaluate differences between each experimental condition (KD and C109S) and WT. Mean ± s.e.m (C) Quantification of mitotic CEP192 foci in PLK4i-treated TP53 −/− MCF-7 cells that lack centrosomes. n = 3, biological replicates, each comprising >30 cells. P values, one-way ANOVA with post hoc Dunnett’s multiple comparisons test to evaluate differences between each experimental condition (KD and C109S) and WT. Mean ± s.e.m (D) Representative images for (C). Scale bars, 5 μm. (E) Representative Sanger sequencing traces for the TRIM37 locus in parental TP53 −/− MCF-7 cells subjected to TRIM37 knockdown (KD) via shRNA, and CRISPR–Cas9 edited TRIM37 C109S KI cells. The mutation (TGT>TCT) leading to the C109S residue substitution is denoted by an asterisk. Silent mutations introduced to prevent re-editing are highlighted.

Article Snippet: Following electrophoresis, proteins were transferred onto nitrocellulose membranes using a Mini Trans-Blot Cell (BioRad) wet transfer system and subsequently probed with the following primary antibodies: TRIM37 (rabbit, Bethyl, A301-174A, 1:1000), HA (rat; Roche, ROAHAHA; 1:1000), β-actin (mouse, Santa Cruz Biotechnology, sc-4778, 1:1000), CEP192 (rabbit, raised against CEP192 residues 1–211, home-made , 1:1000), CNTROB (rabbit, Atlas Antibodies, HPA023319, 1:1000), SAS-6 (mouse, Santa Cruz Biotechnology, sc-81431, 1:1000), vinculin (mouse, Santa Cruz Biotechnology, sc-73614, 1:1000), GAPDH (mouse, Santa Cruz Biotechnology, sc-47724, 1:1000), TRIM37 (rabbit, Cell Signaling, #96167, 1:1000, see ), and mCherry (rabbit, Abcam, ab167453, 1:4000).

Techniques: Western Blot, Knockdown, shRNA, Mutagenesis, Control, Sequencing, CRISPR, Residue

(A) Top, schematic of the TRIM37 G322V -mNeonGreen-CRY2 optogenetic fusion construct. The star denotes the TRAF domain mutation (G322V). Bottom, illustration of the blue light (BL)-activated optogenetic system enabling TRIM37 clustering independent of binding to a centrosome substrate. (B) Representative time-lapse images of RPE-1 cells expressing the optogenetic construct detailed in (A) incubated in the presence or absence of MG132. Timestamps indicate minutes post blue light exposure. Scale bar = 10 µm. (C) Quantification of mNeonGreen fluorescence intensity from (B), with each condition comprising >30 cells. Mean ± s.d. (D) RPE-1 cells expressing optogenetic constructs detailed in (A) were incubated with or without doxycycline (Dox) and MG132 (10 μM) in the absence or presence blue light for 3 h before immunoblotting for the indicated proteins. Higher molecular weight (HMW) TRIM37 species were prominently formed only in MG132 and BL-stimulated conditions and are indicated with an arrow. Ponceau-stained blot indicates loading. Representative data; n = 3 biological replicates. exp, exposure.

Journal: bioRxiv

Article Title: Mesoscale regulation of MTOCs by the E3 ligase TRIM37

doi: 10.1101/2024.10.09.617407

Figure Lengend Snippet: (A) Top, schematic of the TRIM37 G322V -mNeonGreen-CRY2 optogenetic fusion construct. The star denotes the TRAF domain mutation (G322V). Bottom, illustration of the blue light (BL)-activated optogenetic system enabling TRIM37 clustering independent of binding to a centrosome substrate. (B) Representative time-lapse images of RPE-1 cells expressing the optogenetic construct detailed in (A) incubated in the presence or absence of MG132. Timestamps indicate minutes post blue light exposure. Scale bar = 10 µm. (C) Quantification of mNeonGreen fluorescence intensity from (B), with each condition comprising >30 cells. Mean ± s.d. (D) RPE-1 cells expressing optogenetic constructs detailed in (A) were incubated with or without doxycycline (Dox) and MG132 (10 μM) in the absence or presence blue light for 3 h before immunoblotting for the indicated proteins. Higher molecular weight (HMW) TRIM37 species were prominently formed only in MG132 and BL-stimulated conditions and are indicated with an arrow. Ponceau-stained blot indicates loading. Representative data; n = 3 biological replicates. exp, exposure.

Article Snippet: Following electrophoresis, proteins were transferred onto nitrocellulose membranes using a Mini Trans-Blot Cell (BioRad) wet transfer system and subsequently probed with the following primary antibodies: TRIM37 (rabbit, Bethyl, A301-174A, 1:1000), HA (rat; Roche, ROAHAHA; 1:1000), β-actin (mouse, Santa Cruz Biotechnology, sc-4778, 1:1000), CEP192 (rabbit, raised against CEP192 residues 1–211, home-made , 1:1000), CNTROB (rabbit, Atlas Antibodies, HPA023319, 1:1000), SAS-6 (mouse, Santa Cruz Biotechnology, sc-81431, 1:1000), vinculin (mouse, Santa Cruz Biotechnology, sc-73614, 1:1000), GAPDH (mouse, Santa Cruz Biotechnology, sc-47724, 1:1000), TRIM37 (rabbit, Cell Signaling, #96167, 1:1000, see ), and mCherry (rabbit, Abcam, ab167453, 1:4000).

Techniques: Construct, Mutagenesis, Binding Assay, Expressing, Incubation, Fluorescence, Western Blot, Molecular Weight, Staining

(A) Left, schematic depicts the blue light (BL)-triggered optogenetic system designed to cluster TRIM37’s cognate centrosomal substrates, enabling the investigation of TRIM37 recognition and activation requirements. Right, schematic of constructs used in the optogenetic experiments, including mNeonGreen-tagged TRIM37 for visualizing recruitment to centrosomal substrates, mCherry-CRY2 fused to Centrobin’s C-terminal unstructured region (residues 567-836) and a mCherry-CRY2 control. (B) Representative time-lapse images of RPE-1 TRIM37 -/- cells integrated with optogenetic constructs detailed in (A), incubated with or without doxycycline (Dox), in the absence or presence blue light. Timestamps indicate minutes post blue light exposure. Representative data; n = 3 biological replicates. Scale bar = 10 µm. (C) Quantification of mCherry fluorescence intensity from (B), with each condition comprising >30 cells. Mean ± s.d. (D) RPE-1 TRIM37 -/- cells integrated with optogenetic constructs detailed in (A) were incubated with or without doxycycline (Dox), in the absence or presence blue light for 3 h prior to immunoblotting for the indicated proteins. GAPDH, loading control. Representative data; n = 3 biological replicates. (E) Representative time-lapse images of RPE-1 TRIM37 -/- cells integrated with optogenetic constructs and co-expressing different TRIM37 mutants with or without MG132 (10 μM) in the absence or presence blue light. Timestamps indicate minutes post blue light exposure. Representative data; n = 3 biological replicates. Scale bar = 10 µm. mCh, mCherry. (F) Quantification of mCherry fluorescence intensity from (E), with each condition comprising >30 cells. Mean ± s.d. (G) RPE-1 TRIM37 -/- cells expressing indicated optogenetic constructs and different TRIM37 mutants were treated with or without MG132 (10 μM) in the absence or presence of blue light for 3 h prior to immunoblotting for the indicated proteins. GAPDH, loading control. Representative data; n = 3 biological replicates.

Journal: bioRxiv

Article Title: Mesoscale regulation of MTOCs by the E3 ligase TRIM37

doi: 10.1101/2024.10.09.617407

Figure Lengend Snippet: (A) Left, schematic depicts the blue light (BL)-triggered optogenetic system designed to cluster TRIM37’s cognate centrosomal substrates, enabling the investigation of TRIM37 recognition and activation requirements. Right, schematic of constructs used in the optogenetic experiments, including mNeonGreen-tagged TRIM37 for visualizing recruitment to centrosomal substrates, mCherry-CRY2 fused to Centrobin’s C-terminal unstructured region (residues 567-836) and a mCherry-CRY2 control. (B) Representative time-lapse images of RPE-1 TRIM37 -/- cells integrated with optogenetic constructs detailed in (A), incubated with or without doxycycline (Dox), in the absence or presence blue light. Timestamps indicate minutes post blue light exposure. Representative data; n = 3 biological replicates. Scale bar = 10 µm. (C) Quantification of mCherry fluorescence intensity from (B), with each condition comprising >30 cells. Mean ± s.d. (D) RPE-1 TRIM37 -/- cells integrated with optogenetic constructs detailed in (A) were incubated with or without doxycycline (Dox), in the absence or presence blue light for 3 h prior to immunoblotting for the indicated proteins. GAPDH, loading control. Representative data; n = 3 biological replicates. (E) Representative time-lapse images of RPE-1 TRIM37 -/- cells integrated with optogenetic constructs and co-expressing different TRIM37 mutants with or without MG132 (10 μM) in the absence or presence blue light. Timestamps indicate minutes post blue light exposure. Representative data; n = 3 biological replicates. Scale bar = 10 µm. mCh, mCherry. (F) Quantification of mCherry fluorescence intensity from (E), with each condition comprising >30 cells. Mean ± s.d. (G) RPE-1 TRIM37 -/- cells expressing indicated optogenetic constructs and different TRIM37 mutants were treated with or without MG132 (10 μM) in the absence or presence of blue light for 3 h prior to immunoblotting for the indicated proteins. GAPDH, loading control. Representative data; n = 3 biological replicates.

Article Snippet: Following electrophoresis, proteins were transferred onto nitrocellulose membranes using a Mini Trans-Blot Cell (BioRad) wet transfer system and subsequently probed with the following primary antibodies: TRIM37 (rabbit, Bethyl, A301-174A, 1:1000), HA (rat; Roche, ROAHAHA; 1:1000), β-actin (mouse, Santa Cruz Biotechnology, sc-4778, 1:1000), CEP192 (rabbit, raised against CEP192 residues 1–211, home-made , 1:1000), CNTROB (rabbit, Atlas Antibodies, HPA023319, 1:1000), SAS-6 (mouse, Santa Cruz Biotechnology, sc-81431, 1:1000), vinculin (mouse, Santa Cruz Biotechnology, sc-73614, 1:1000), GAPDH (mouse, Santa Cruz Biotechnology, sc-47724, 1:1000), TRIM37 (rabbit, Cell Signaling, #96167, 1:1000, see ), and mCherry (rabbit, Abcam, ab167453, 1:4000).

Techniques: Activation Assay, Construct, Control, Incubation, Fluorescence, Western Blot, Expressing

(A) Model illustrating how TRIM37 regulates MTOCs through substrate-templated higher-order assembly, demonstrated here using centrosomes, highlighting a conserved mechanism reminiscent of TRIM5’s role in HIV capsid restriction.

Journal: bioRxiv

Article Title: Mesoscale regulation of MTOCs by the E3 ligase TRIM37

doi: 10.1101/2024.10.09.617407

Figure Lengend Snippet: (A) Model illustrating how TRIM37 regulates MTOCs through substrate-templated higher-order assembly, demonstrated here using centrosomes, highlighting a conserved mechanism reminiscent of TRIM5’s role in HIV capsid restriction.

Article Snippet: Following electrophoresis, proteins were transferred onto nitrocellulose membranes using a Mini Trans-Blot Cell (BioRad) wet transfer system and subsequently probed with the following primary antibodies: TRIM37 (rabbit, Bethyl, A301-174A, 1:1000), HA (rat; Roche, ROAHAHA; 1:1000), β-actin (mouse, Santa Cruz Biotechnology, sc-4778, 1:1000), CEP192 (rabbit, raised against CEP192 residues 1–211, home-made , 1:1000), CNTROB (rabbit, Atlas Antibodies, HPA023319, 1:1000), SAS-6 (mouse, Santa Cruz Biotechnology, sc-81431, 1:1000), vinculin (mouse, Santa Cruz Biotechnology, sc-73614, 1:1000), GAPDH (mouse, Santa Cruz Biotechnology, sc-47724, 1:1000), TRIM37 (rabbit, Cell Signaling, #96167, 1:1000, see ), and mCherry (rabbit, Abcam, ab167453, 1:4000).

Techniques:

A The mRNA and protein expressions of TRIM37 in KIFC1-overexpressed HEC-1A and KIFC1-knockdown Ishikawa cells. B The mRNA and protein expressions of TRIM37 in PLK4-overexpressed and KIFC1-knockdown EC cell lines (HEC-1A and Ishikawa). C Results of the ubiquitination level of PLK4 in HEK293T cells after KIFC1 or TRIM37 overexpression detected by ubiquitination assay. D The mRNA expressions of KIFC1, TRIM37, and PLK4 in TRIM37- and KIFC1-overexpressed EC cell lines (HEC-1A and Ishikawa). E CCK8 assay in TRIM37- and KIFC1-overexpressed EC cell lines (HEC-1A and Ishikawa). F Colony formation assay in TRIM37- and KIFC1-overexpressed EC cell lines (HEC-1A and Ishikawa). G Transwell assay and quantitative analysis of migratory and invasive abilities of TRIM37- and KIFC1-overexpressed EC cell lines (HEC-1A and Ishikawa) (scale bar: 100 μm). H The protein expressions of KIFC1, TRIM37, and PLK4 in TRIM37- and KIFC1-overexpressed EC cell lines (HEC-1A and Ishikawa). * P < 0.05, ** P < 0.01, # P < 0.05, ## P < 0.01.

Journal: Cell Death Discovery

Article Title: KIFC1 depends on TRIM37-mediated ubiquitination of PLK4 to promote centrosome amplification in endometrial cancer

doi: 10.1038/s41420-024-02190-1

Figure Lengend Snippet: A The mRNA and protein expressions of TRIM37 in KIFC1-overexpressed HEC-1A and KIFC1-knockdown Ishikawa cells. B The mRNA and protein expressions of TRIM37 in PLK4-overexpressed and KIFC1-knockdown EC cell lines (HEC-1A and Ishikawa). C Results of the ubiquitination level of PLK4 in HEK293T cells after KIFC1 or TRIM37 overexpression detected by ubiquitination assay. D The mRNA expressions of KIFC1, TRIM37, and PLK4 in TRIM37- and KIFC1-overexpressed EC cell lines (HEC-1A and Ishikawa). E CCK8 assay in TRIM37- and KIFC1-overexpressed EC cell lines (HEC-1A and Ishikawa). F Colony formation assay in TRIM37- and KIFC1-overexpressed EC cell lines (HEC-1A and Ishikawa). G Transwell assay and quantitative analysis of migratory and invasive abilities of TRIM37- and KIFC1-overexpressed EC cell lines (HEC-1A and Ishikawa) (scale bar: 100 μm). H The protein expressions of KIFC1, TRIM37, and PLK4 in TRIM37- and KIFC1-overexpressed EC cell lines (HEC-1A and Ishikawa). * P < 0.05, ** P < 0.01, # P < 0.05, ## P < 0.01.

Article Snippet: The blots were then blocked in 5% nonfat dry milk solution (Beyotime Biotechnology, Shanghai) at room temperature for 1 h. Subsequently, the blots were incubated with the following primary antibodies: KIFC1 (1:1000, No. 20790-1-AP, Proteintech), γ-tubulin (1:5000, No. 66320-1-Ig, Proteintech), p-H3 (S10) (1:1000, #9701, Cell Signaling Technology), Cyclin A2 (1:1000, #67955, Cell Signaling Technology), Cyclin B1 (1:1000, #4138, Cell Signaling Technology), CDK1 (1:10000, No. 10122-1-AP, Proteintech), CDC2 (1:1000, #77055, Cell Signaling Technology), PLK4 (1:1000, No. 12952-1-AP, Proteintech), TRIM37 (1:15.00, No. 13037-1-AP, Proteintech), Ub (1:1000, ab134953, Abcam), Myc (1:1000, ab32, Abcam), HA (1:1000, ab1424, Abcam), His (1:1000, ab18184, Abcam) and GAPDH (1:10000, ET1601-4, Huabio) at 4 °C overnight.

Techniques: Knockdown, Over Expression, Ubiquitin Assay, CCK-8 Assay, Colony Assay, Transwell Assay

A General images of mice and tumors in the subcutaneous xenograft of HEC-1A cells after TRIM37 and KIFC1 overexpression. B The tumor volume and weight. C Representative H & E staining and IHC staining of Ki-67, KIFC1, TRIM37, and PLK4 in tumors (scale bar: 200 μm for left; 100 μm for right). D Representative TUNEL images of tumors (scale bar: 200 μm for left; 100 μm for right). E Representative H & E staining images of lung tissues (scale bar: 200 μm for left; 100 μm for right); F Fluorescence images (left) and quantitative analysis (right) of mouse lung metastasis. * P < 0.05, ** P < 0.01, # P < 0.05, ## P < 0.01.

Journal: Cell Death Discovery

Article Title: KIFC1 depends on TRIM37-mediated ubiquitination of PLK4 to promote centrosome amplification in endometrial cancer

doi: 10.1038/s41420-024-02190-1

Figure Lengend Snippet: A General images of mice and tumors in the subcutaneous xenograft of HEC-1A cells after TRIM37 and KIFC1 overexpression. B The tumor volume and weight. C Representative H & E staining and IHC staining of Ki-67, KIFC1, TRIM37, and PLK4 in tumors (scale bar: 200 μm for left; 100 μm for right). D Representative TUNEL images of tumors (scale bar: 200 μm for left; 100 μm for right). E Representative H & E staining images of lung tissues (scale bar: 200 μm for left; 100 μm for right); F Fluorescence images (left) and quantitative analysis (right) of mouse lung metastasis. * P < 0.05, ** P < 0.01, # P < 0.05, ## P < 0.01.

Article Snippet: The blots were then blocked in 5% nonfat dry milk solution (Beyotime Biotechnology, Shanghai) at room temperature for 1 h. Subsequently, the blots were incubated with the following primary antibodies: KIFC1 (1:1000, No. 20790-1-AP, Proteintech), γ-tubulin (1:5000, No. 66320-1-Ig, Proteintech), p-H3 (S10) (1:1000, #9701, Cell Signaling Technology), Cyclin A2 (1:1000, #67955, Cell Signaling Technology), Cyclin B1 (1:1000, #4138, Cell Signaling Technology), CDK1 (1:10000, No. 10122-1-AP, Proteintech), CDC2 (1:1000, #77055, Cell Signaling Technology), PLK4 (1:1000, No. 12952-1-AP, Proteintech), TRIM37 (1:15.00, No. 13037-1-AP, Proteintech), Ub (1:1000, ab134953, Abcam), Myc (1:1000, ab32, Abcam), HA (1:1000, ab1424, Abcam), His (1:1000, ab18184, Abcam) and GAPDH (1:10000, ET1601-4, Huabio) at 4 °C overnight.

Techniques: Over Expression, Staining, Immunohistochemistry, TUNEL Assay, Fluorescence

A The mRNA and protein expressions of TRIM37 in KIFC1-overexpressed HEC-1A and KIFC1-knockdown Ishikawa cells. B The mRNA and protein expressions of TRIM37 in PLK4-overexpressed and KIFC1-knockdown EC cell lines (HEC-1A and Ishikawa). C Results of the ubiquitination level of PLK4 in HEK293T cells after KIFC1 or TRIM37 overexpression detected by ubiquitination assay. D The mRNA expressions of KIFC1, TRIM37, and PLK4 in TRIM37- and KIFC1-overexpressed EC cell lines (HEC-1A and Ishikawa). E CCK8 assay in TRIM37- and KIFC1-overexpressed EC cell lines (HEC-1A and Ishikawa). F Colony formation assay in TRIM37- and KIFC1-overexpressed EC cell lines (HEC-1A and Ishikawa). G Transwell assay and quantitative analysis of migratory and invasive abilities of TRIM37- and KIFC1-overexpressed EC cell lines (HEC-1A and Ishikawa) (scale bar: 100 μm). H The protein expressions of KIFC1, TRIM37, and PLK4 in TRIM37- and KIFC1-overexpressed EC cell lines (HEC-1A and Ishikawa). * P < 0.05, ** P < 0.01, # P < 0.05, ## P < 0.01.

Journal: Cell Death Discovery

Article Title: KIFC1 depends on TRIM37-mediated ubiquitination of PLK4 to promote centrosome amplification in endometrial cancer

doi: 10.1038/s41420-024-02190-1

Figure Lengend Snippet: A The mRNA and protein expressions of TRIM37 in KIFC1-overexpressed HEC-1A and KIFC1-knockdown Ishikawa cells. B The mRNA and protein expressions of TRIM37 in PLK4-overexpressed and KIFC1-knockdown EC cell lines (HEC-1A and Ishikawa). C Results of the ubiquitination level of PLK4 in HEK293T cells after KIFC1 or TRIM37 overexpression detected by ubiquitination assay. D The mRNA expressions of KIFC1, TRIM37, and PLK4 in TRIM37- and KIFC1-overexpressed EC cell lines (HEC-1A and Ishikawa). E CCK8 assay in TRIM37- and KIFC1-overexpressed EC cell lines (HEC-1A and Ishikawa). F Colony formation assay in TRIM37- and KIFC1-overexpressed EC cell lines (HEC-1A and Ishikawa). G Transwell assay and quantitative analysis of migratory and invasive abilities of TRIM37- and KIFC1-overexpressed EC cell lines (HEC-1A and Ishikawa) (scale bar: 100 μm). H The protein expressions of KIFC1, TRIM37, and PLK4 in TRIM37- and KIFC1-overexpressed EC cell lines (HEC-1A and Ishikawa). * P < 0.05, ** P < 0.01, # P < 0.05, ## P < 0.01.

Article Snippet: The antibodies anti-KIFC1 (1:200, No. 20790-1-AP, Proteintech), anti-PLK4 (1:200, No. 12952-1-AP, Proteintech), anti-TRIM37 (1:200, No. 13037-1-AP, Proteintech) and anti-Ki-67 (1:200, 27309-1-AP, Proteintech) were used for IHC staining.

Techniques: Knockdown, Over Expression, Ubiquitin Assay, CCK-8 Assay, Colony Assay, Transwell Assay

A General images of mice and tumors in the subcutaneous xenograft of HEC-1A cells after TRIM37 and KIFC1 overexpression. B The tumor volume and weight. C Representative H & E staining and IHC staining of Ki-67, KIFC1, TRIM37, and PLK4 in tumors (scale bar: 200 μm for left; 100 μm for right). D Representative TUNEL images of tumors (scale bar: 200 μm for left; 100 μm for right). E Representative H & E staining images of lung tissues (scale bar: 200 μm for left; 100 μm for right); F Fluorescence images (left) and quantitative analysis (right) of mouse lung metastasis. * P < 0.05, ** P < 0.01, # P < 0.05, ## P < 0.01.

Journal: Cell Death Discovery

Article Title: KIFC1 depends on TRIM37-mediated ubiquitination of PLK4 to promote centrosome amplification in endometrial cancer

doi: 10.1038/s41420-024-02190-1

Figure Lengend Snippet: A General images of mice and tumors in the subcutaneous xenograft of HEC-1A cells after TRIM37 and KIFC1 overexpression. B The tumor volume and weight. C Representative H & E staining and IHC staining of Ki-67, KIFC1, TRIM37, and PLK4 in tumors (scale bar: 200 μm for left; 100 μm for right). D Representative TUNEL images of tumors (scale bar: 200 μm for left; 100 μm for right). E Representative H & E staining images of lung tissues (scale bar: 200 μm for left; 100 μm for right); F Fluorescence images (left) and quantitative analysis (right) of mouse lung metastasis. * P < 0.05, ** P < 0.01, # P < 0.05, ## P < 0.01.

Article Snippet: The antibodies anti-KIFC1 (1:200, No. 20790-1-AP, Proteintech), anti-PLK4 (1:200, No. 12952-1-AP, Proteintech), anti-TRIM37 (1:200, No. 13037-1-AP, Proteintech) and anti-Ki-67 (1:200, 27309-1-AP, Proteintech) were used for IHC staining.

Techniques: Over Expression, Staining, Immunohistochemistry, TUNEL Assay, Fluorescence

PCR primer sequences

Journal: Journal of Cancer Research and Clinical Oncology

Article Title: LncRNA HOXC-AS3 accelerates malignant proliferation of cervical cancer cells via stabilizing KDM5B

doi: 10.1007/s00432-024-05799-y

Figure Lengend Snippet: PCR primer sequences

Article Snippet: After the blockade with 5% defatted milk at room temperature for 2 h, membranes were incubated with the following primary antibodies: KDM5B (ab181089, 1:1000, Abcam, Cambridge, MA, USA), TRIM37 (ab264190, 1:2000, Abcam, Cambridge, MA, USA), and GAPDH (ab9485, 1:2500, Abcam, Cambridge, MA, USA) at 4 ℃ overnight, and with goat anti-rabbit immunoglobulin G (horseradish peroxidase) antibody (ab205718, 1:2000, Abcam, Cambridge, MA, USA) at room temperature for 1 h after being washed with PBS thrice.

Techniques: Sequencing

HOXC-AS3 diminishes KDM5B ubiquitination via binding to KDM5B. A , B KDM5B expression levels in CC tissues ( N = 45; representative bands) and CC cells were detected via WB assay. C E3 ubiquitin ligases that can regulate KDM5B were predicted via the Ubibrowser database. CaSki cells were transfected with TRIM37 siRNA (si-TRIM37) with NC siRNA (si-NC) as the negative control. D , E TRIM37 expression levels in CaSki cells were detected via RT-qPCR and WB assays. F The ubiquitination levels of KDM5B cells in each group were detected via the ubiquitination assay. Cell experiments were independently repeated 3 times. Data were shown as mean ± standard deviation; data comparisons in panels A , B (right), and E were tested via the t -test; data comparisons in panels B , D were tested via one-way ANOVA, followed by Tukey’s multiple comparison test. * p < 0.05; ** p < 0.01

Journal: Journal of Cancer Research and Clinical Oncology

Article Title: LncRNA HOXC-AS3 accelerates malignant proliferation of cervical cancer cells via stabilizing KDM5B

doi: 10.1007/s00432-024-05799-y

Figure Lengend Snippet: HOXC-AS3 diminishes KDM5B ubiquitination via binding to KDM5B. A , B KDM5B expression levels in CC tissues ( N = 45; representative bands) and CC cells were detected via WB assay. C E3 ubiquitin ligases that can regulate KDM5B were predicted via the Ubibrowser database. CaSki cells were transfected with TRIM37 siRNA (si-TRIM37) with NC siRNA (si-NC) as the negative control. D , E TRIM37 expression levels in CaSki cells were detected via RT-qPCR and WB assays. F The ubiquitination levels of KDM5B cells in each group were detected via the ubiquitination assay. Cell experiments were independently repeated 3 times. Data were shown as mean ± standard deviation; data comparisons in panels A , B (right), and E were tested via the t -test; data comparisons in panels B , D were tested via one-way ANOVA, followed by Tukey’s multiple comparison test. * p < 0.05; ** p < 0.01

Article Snippet: After the blockade with 5% defatted milk at room temperature for 2 h, membranes were incubated with the following primary antibodies: KDM5B (ab181089, 1:1000, Abcam, Cambridge, MA, USA), TRIM37 (ab264190, 1:2000, Abcam, Cambridge, MA, USA), and GAPDH (ab9485, 1:2500, Abcam, Cambridge, MA, USA) at 4 ℃ overnight, and with goat anti-rabbit immunoglobulin G (horseradish peroxidase) antibody (ab205718, 1:2000, Abcam, Cambridge, MA, USA) at room temperature for 1 h after being washed with PBS thrice.

Techniques: Binding Assay, Expressing, Transfection, Negative Control, Quantitative RT-PCR, Ubiquitin Assay, Standard Deviation, Comparison

HOXC-AS3 interferes with the interaction between TRIM37 and KDM5B. A The interaction between KDM5B and TRIM37 was analyzed via the Co-IP assay. B The cellular co-location of KDM5B and TRIM37 was determined via the immunofluorescence assay. C The interaction between KDM5B and TRIM37 was analyzed via the Co-IP assay. Cell experiments were independently repeated 3 times

Journal: Journal of Cancer Research and Clinical Oncology

Article Title: LncRNA HOXC-AS3 accelerates malignant proliferation of cervical cancer cells via stabilizing KDM5B

doi: 10.1007/s00432-024-05799-y

Figure Lengend Snippet: HOXC-AS3 interferes with the interaction between TRIM37 and KDM5B. A The interaction between KDM5B and TRIM37 was analyzed via the Co-IP assay. B The cellular co-location of KDM5B and TRIM37 was determined via the immunofluorescence assay. C The interaction between KDM5B and TRIM37 was analyzed via the Co-IP assay. Cell experiments were independently repeated 3 times

Article Snippet: After the blockade with 5% defatted milk at room temperature for 2 h, membranes were incubated with the following primary antibodies: KDM5B (ab181089, 1:1000, Abcam, Cambridge, MA, USA), TRIM37 (ab264190, 1:2000, Abcam, Cambridge, MA, USA), and GAPDH (ab9485, 1:2500, Abcam, Cambridge, MA, USA) at 4 ℃ overnight, and with goat anti-rabbit immunoglobulin G (horseradish peroxidase) antibody (ab205718, 1:2000, Abcam, Cambridge, MA, USA) at room temperature for 1 h after being washed with PBS thrice.

Techniques: Co-Immunoprecipitation Assay, Immunofluorescence

HOXC-AS3 promoted malignant proliferation of CC cells via regulating KDM5B. HOXC-AS3 directly bound to KDM5B, which inhibited TRIM37-mediated KDM5B ubiquitination and upregulated KDM5B protein levels, promoting malignant proliferation of CC cells

Journal: Journal of Cancer Research and Clinical Oncology

Article Title: LncRNA HOXC-AS3 accelerates malignant proliferation of cervical cancer cells via stabilizing KDM5B

doi: 10.1007/s00432-024-05799-y

Figure Lengend Snippet: HOXC-AS3 promoted malignant proliferation of CC cells via regulating KDM5B. HOXC-AS3 directly bound to KDM5B, which inhibited TRIM37-mediated KDM5B ubiquitination and upregulated KDM5B protein levels, promoting malignant proliferation of CC cells

Article Snippet: After the blockade with 5% defatted milk at room temperature for 2 h, membranes were incubated with the following primary antibodies: KDM5B (ab181089, 1:1000, Abcam, Cambridge, MA, USA), TRIM37 (ab264190, 1:2000, Abcam, Cambridge, MA, USA), and GAPDH (ab9485, 1:2500, Abcam, Cambridge, MA, USA) at 4 ℃ overnight, and with goat anti-rabbit immunoglobulin G (horseradish peroxidase) antibody (ab205718, 1:2000, Abcam, Cambridge, MA, USA) at room temperature for 1 h after being washed with PBS thrice.

Techniques: