Journal: Cancers

Article Title: MYC Regulates a DNA Repair Gene Expression Program in Small Cell Carcinoma of the Ovary, Hypercalcemic Type

doi: 10.3390/cancers17132255

Figure Lengend Snippet: Depletion of MYC in BIN-67 cells. ( a ) Engineered BIN-67 (clone 1) cells were treated with DMSO, 500 nM dTAG V -1-NEG (NEG), or 500 nM dTAG V -1 (V1) for 24 h to deplete MYC. GAPDH is included as a loading control. SNF5 and BAF155 are SWI/SNF subunits. Uncropped Western blots are included in . ( b ) Engineered BIN-67 (clone 9) cells were treated as in ( a ) for 24 h to deplete MYC. Similar controls are included. Uncropped Western blots are included in . ( c ) Engineered BIN-67 (clone 1) cells were plated with 500 nM NEG, 500 nM V1, or DMSO and then allowed to grow for five days before recounting. The fold change in cell number was determined using the total cell number obtained on day five as compared to the day of plating. (The error bars are the standard error of the mean, unpaired student’s t -test ** p = 0.0019, n = 3 biological replicates). ( d ) The engineered BIN-67 (clone 9) cells were plated as in ( c ), and the fold change was calculated and graphed. (The error bars are the standard error of the mean, unpaired student t -test ** p = 0.0089, n = 4 biological replicates). ( e ) The engineered BIN-67 (clone 1) cells were plated with 500 nM NEG, 500 nM V1, or DMSO. After five days, a cell cycle analysis was performed, and the data were quantified for each phase, as shown. (The error bars are the standard error of the mean, one-way ANOVA analysis Sub-G1 ** p = 0.0030, G2/M ** p = 0.0018, n.s. = not significant, n = 4 biological replicates).

Article Snippet: Approximately 2.0 × 10 6 cells were treated with either dimethyl sulfoxide (DMSO), 500 nM dTAG V -1-NEG (Tocris, 6915, Minneapolis, MN, USA), or 500 nM dTAG V -1 (Tocris, 6914) for 24 h before the cells were washed twice with phosphate-buffered saline (PBS), containing 137 mM NaCl, 2.7 mM KCl, and 10 mM phosphate (VWR, 76371-734, Radnor, PA, USA) and pelleted.

Techniques: Control, Western Blot, Cell Cycle Assay

Journal: Cancers

Article Title: MYC Regulates a DNA Repair Gene Expression Program in Small Cell Carcinoma of the Ovary, Hypercalcemic Type

doi: 10.3390/cancers17132255

Figure Lengend Snippet: Depletion of MYC impacts the expression of diverse sets of genes. ( a ) The magnitude and significance of gene expression changes resulting from an RNA-seq analysis of engineered BIN-67 cells treated with DMSO or 500 nM dTAG V -1-NEG (NEG) for 24 h. The grey lines denote a false discovery rate (FDR) of 0.05 and a fold change of 1.5. ( b ) A volcano plot showing the magnitude and significance of gene expression changes from an RNA-seq analysis of engineered BIN-67 cells treated with 500 nM NEG or dTAG V -1 (V1). The blue dots indicate genes that decrease significantly in expression (FDR < 0.05) with a fold change less than −1.5. The red dots indicate genes that increase significantly (FDR < 0.05) in expression and have a fold change that is higher than 1.5. ( c ) A heatmap depicting z-score-normalized read counts for genes that significantly changed in expression (FDR < 0.05) following treatment with V1. The data for each replicate is included. ( d ) A gene ontology analysis was performed using genes that decrease in expression following MYC depletion, as determined in the V1 vs. NEG analysis (FDR < 0.05, fold change < −1.5). The analysis was performed using ShinyGO 0.82 and “GO Biological Process”. ( e ) A gene set enrichment analysis (GSEA) was performed by testing the differentially expressed genes (V1 vs. NEG) against MSigDB hallmark datasets [ , ]. The loss of MYC causes an upregulation of genes related to coagulation and epithelial mesenchymal transition. ( f ) GSEA was performed as in ( e ). The loss of MYC downregulates genes related to E2F targets, DNA repair, G2/M checkpoint, and oxidative phosphorylation. The complete GSEA results are in .

Article Snippet: Approximately 2.0 × 10 6 cells were treated with either dimethyl sulfoxide (DMSO), 500 nM dTAG V -1-NEG (Tocris, 6915, Minneapolis, MN, USA), or 500 nM dTAG V -1 (Tocris, 6914) for 24 h before the cells were washed twice with phosphate-buffered saline (PBS), containing 137 mM NaCl, 2.7 mM KCl, and 10 mM phosphate (VWR, 76371-734, Radnor, PA, USA) and pelleted.

Techniques: Expressing, Gene Expression, RNA Sequencing, Coagulation, Phospho-proteomics

Journal: Cancers

Article Title: MYC Regulates a DNA Repair Gene Expression Program in Small Cell Carcinoma of the Ovary, Hypercalcemic Type

doi: 10.3390/cancers17132255

Figure Lengend Snippet: Gene regulation by chromatin-bound MYC. ( a ) Magnitude of change in expression for genes bound by MYC that go up (red) or down (blue) following depletion of MYC in BIN-67 cells. MYC-bound genes are defined as a gene with an MYC binding site localized within 1 kb of the annotated TSS. ( b ) Gene ontology analysis of MYC-bound genes that decrease in expression following the depletion of MYC using DAVID Bioinformatics [ , ]. The number next to each bar signifies the number of genes in that category. ( c ) A KEGG pathway analysis was performed on MYC-bound genes that decrease in expression following MYC depletion. An analysis was performed using ShinyGO 0.82 and the “KEGG pathway database” . ( d ) A Venn diagram comparing the number of significantly downregulated genes in the V1 vs. NEG RNA-seq analysis with genes annotated to MYC binding sites and a curated list of DNA repair genes from . The Venn diagram was generated using https://molbiotools.com/listcompare.php (Accessed on 3 March 2025). ( e ) Top: heatmap showing the magnitude of gene expression changes for the 27 MYC-bound DNA repair genes from ( d ). Genes marked with solid yellow are bound by MYC but have no detectable change in gene expression. Bottom: heatmap showing the magnitude of gene expression changes for the 84 non-MYC-bound DNA repair genes from ( d ). ( f ) Western blot showing the levels of MYC, ATR, and RAD51 in BIN-67 (clone 1) cells treated with DMSO, 500 nM NEG, or 500 nM V1 for 24 h. GAPDH serves as a loading control. ( g ) Western blot as described in ( f ) for BIN-67 (clone 9) cells treated similarly. ( h ) Western blot as described in ( f ) for engineered SNF5-null G401 cells treated similarly. Uncropped Western blots are included in .

Article Snippet: Approximately 2.0 × 10 6 cells were treated with either dimethyl sulfoxide (DMSO), 500 nM dTAG V -1-NEG (Tocris, 6915, Minneapolis, MN, USA), or 500 nM dTAG V -1 (Tocris, 6914) for 24 h before the cells were washed twice with phosphate-buffered saline (PBS), containing 137 mM NaCl, 2.7 mM KCl, and 10 mM phosphate (VWR, 76371-734, Radnor, PA, USA) and pelleted.

Techniques: Expressing, Binding Assay, RNA Sequencing, Generated, Gene Expression, Western Blot, Control