Journal: Scientific reports
Article Title: Focused CRISPR-Cas9 genetic screening reveals USO1 as a vulnerability in B-cell acute lymphoblastic leukemia.
doi: 10.1038/s41598-021-92448-w
Figure Lengend Snippet: Figure 2. Dependence of RBP gene expression on MLL-AF4 translocation. (A–C) Effect of I-BET 151 and MI-503 treatment on mRNA expression levels of USO1,EPRS and EIF3E, measured by RT-qPCR, in SEM (A), RS4;11 (B), and NALM6 (C). The cells were treated with increasing concentrations of I-BET151 (DMSO only, 0.5, 1 and 2 μM) or with increasing concentrations of the menin inhibitor, MI-503 (DMSO only, 0.12, 0.25, and 0.5 μM). RT-qPCR was performed with an optimized set of primers, normalized to 18S, and then represented as fold-change from vehicle-treated control. D. Western blot analysis of murine bone marrow cells with and without transduction with MLL-Af4 (WT versus MLL-Af4), for MLL1 (top), USO1 (middle) and β-actin (lower). (E) UCSC genome browser shot of the USO1 locus showing the MLL-AF4 ChIP site(s), as identified from the ChIP-Seq data from Lin et al.21, in a gene expression regulatory region; Courtesy: UCSC Genome Browser. Shown are the H3K27Ac track in hematopoietic K562 cells (Blue), and MLL-AF4 binding sites represented as a grayscale score, with black indicating the highest score/highest number of reads from the dataset. (F) Chromatin immunoprecipitation with indicated antibodies (MLL1, AF4, and RNA Pol II), followed by qPCR (ChIP-qPCR) analysis for quantitation of bound USO1 promoter/regulatory region to MLL1 and AF4 pulldown samples. Shown is the fold-enrichment for qPCR of the USO1 regulatory site over background (t test; *P < 0.05) (G) SEM cells treated with 1 µM of I-BET151 for 48 h. and subjected to ChIP qPCR with MLL1 and AF4 antibodies as in (F) (t test; *P < 0.05).
Article Snippet: EPRS (#A303-957A), EIF3E (#A302-984A), USO1 (#A304-513A) antibodies were purchased from Bethyl laboratories.
Techniques: Gene Expression, Translocation Assay, Expressing, Quantitative RT-PCR, Control, Western Blot, Transduction, ChIP-sequencing, Binding Assay, Chromatin Immunoprecipitation, ChIP-qPCR, Quantitation Assay
Bethyl is a verified supplier 
