quantigene sample processing kit for cultured cells  (Thermo Fisher)

Journal: eLife

Article Title: Ribosome subunit attrition and activation of the p53–MDM4 axis dominate the response of MLL-rearranged cancer cells to WDR5 WIN site inhibition

doi: 10.7554/eLife.90683

Figure Lengend Snippet: ( A ) Chemical structures of C6 and C16. ( B ) Crystal structures of C6 or C16 bound to the WIN site of WDR5 with electrostatic surfaces mapped (PDB IDs: 6E23 ; 6UCS ). The image shows a close-up view of the WIN site. ( C ) Superimposed WIN site-binding conformations of C6 (green) and C16 (blue). ( D ) Transcript levels as determined by QuantiGene analysis of representative WDR5-bound (color) or non-bound (grayscale) ribosomal protein genes in MV4;11 cells treated with a serial dilution range of either C6 (left) or C16 (right) and relative to DMSO-treated cells (n = 2–3; mean ± SEM). Vertical dashed line indicates either 2 µM C6 (left) or 100 nM C16 (right). ( E ) Number of genes with significantly (false discovery rate [FDR] < 0.05) altered transcript levels following treatment of MV4;11 cells with C6 (2 µM) or C16 (100 nM) for 48 hr, as determined by RNA-Seq (n = 3). See for complete output of RNA-seq analysis. ( F ) Comparison of gene expression changes elicited by C6 (x-axis) and C16 (y-axis), represented as Log2 fold change (FC) compared to DMSO. WDR5-bound genes are colored red. Locations of RPL22L1 and ZMAT3 are indicated. ( G ) Overlap of genes with decreased (left) or increased (right) transcript levels in MV4;11 cells treated with C6 or C16. ( H ) Gene set enrichment analysis (GSEA) showing the distribution of genes suppressed in MV4;11 cells in response to C6 (left) or C16 (right) against the list of all genes bound by WDR5 in those cells . NES, normalized enrichment score. ( I ) Enrichment analysis of genes suppressed (left) or induced (right) by C6 or C16 in MV4;11 cells. KEGG and Hallmark.MSigDB pathways are shown. Fold enrichment of indicated pathways is presented on the x-axis, the number of genes is shown in italics in each bar, and colors represent -Log10 FDR. See for additional GSEA (Hallmark) and over-representation analysis (ORA) (Hallmark) analyses of differentially expressed genes. ( J ) Transcript level changes in WDR5-bound (left) and non-bound (right) RPGs elicited by C6 (top) or C16 (bottom). Figure 1—source data 1. Output of RNA-seq analysis of MV4;11 cells treated with C6/C16. Figure 1—source data 2. GSEA Hallmark and over-representation analysis (ORA) Hallmark enrichment analysis of differentially expressed genes in RNA-seq.

Article Snippet: A custom QuantiGene Plex panel (Thermo Fisher Scientific) was used in conjunction with the QuantiGene Sample Processing Kit for cultured cells (Thermo Fisher Scientific), and QuantiGene Plex Assay kit (Thermo Fisher Scientific) to quantify transcripts following the manufacturer’s instructions.

Techniques: Binding Assay, Serial Dilution, RNA Sequencing Assay, Comparison, Expressing

Journal: eLife

Article Title: Ribosome subunit attrition and activation of the p53–MDM4 axis dominate the response of MLL-rearranged cancer cells to WDR5 WIN site inhibition

doi: 10.7554/eLife.90683

Figure Lengend Snippet: ( A ) Crystal structures of C6 or C16 bound to the WIN Site of WDR5 with electrostatic surfaces mapped (PDB IDs: 6E23 ; 6UCS ). ( B ) Viabilities of MV4;11 cells treated with a serial dilution range of either C6 (left) or C16 (right) for 72 hr, relative to viability of DMSO-treated cells (n = 3; mean ± SEM). ( C ) As in ( B ) but for MOLM13 cells. ( D ) Transcript levels as determined by QuantiGene analysis of representative WDR5-bound (color) or non-bound (grayscale) ribosomal protein genes in MOLM13 cells treated with a serial dilution range of either C6 (left) or C16 (right) and relative to DMSO-treated cells (n = 3; mean ± SEM). Vertical dashed line indicates either 2 µM C6 (left) or 100 nM C16 (right). ( E ) Transformed z-scores of genes with significantly altered transcript levels (RNA-seq) in MV4;11 cells treated with either C6 (2 µM) or C16 (100 nM) for 48 hr, compared to DMSO treatment. ( F ) Volcano plots, showing transcript level alterations in MV4;11 cells treated 48 hr with 2 µM C6 (left) or 100 nM C16 (right) compared to DMSO (n = 3; red indicates false discovery rate [FDR] < 0.05). ( G ) Dispersion plot describing the variance in gene expression for the RNA-seq data in a previous study (left) and this study (right).

Article Snippet: A custom QuantiGene Plex panel (Thermo Fisher Scientific) was used in conjunction with the QuantiGene Sample Processing Kit for cultured cells (Thermo Fisher Scientific), and QuantiGene Plex Assay kit (Thermo Fisher Scientific) to quantify transcripts following the manufacturer’s instructions.

Techniques: Serial Dilution, Transformation Assay, RNA Sequencing Assay, Dispersion, Expressing

Journal: eLife

Article Title: Ribosome subunit attrition and activation of the p53–MDM4 axis dominate the response of MLL-rearranged cancer cells to WDR5 WIN site inhibition

doi: 10.7554/eLife.90683

Figure Lengend Snippet:

Article Snippet: A custom QuantiGene Plex panel (Thermo Fisher Scientific) was used in conjunction with the QuantiGene Sample Processing Kit for cultured cells (Thermo Fisher Scientific), and QuantiGene Plex Assay kit (Thermo Fisher Scientific) to quantify transcripts following the manufacturer’s instructions.

Techniques: Recombinant, Plasmid Preparation, CRISPR, Knock-Out, Protease Inhibitor, Staining, Mass Spectrometry, Transfection, Gene Knockout, Negative Control, Cell Culture, Plex Assay, Bicinchoninic Acid Protein Assay, Western Blot, Sensitive Assay, Cell Viability Assay, Software, Luminex, Real-time Polymerase Chain Reaction, Imaging