Journal: Applied and Environmental Microbiology

Article Title: The Cold-Induced Two-Component System CBO0366/CBO0365 Regulates Metabolic Pathways with Novel Roles in Group I Clostridium botulinum ATCC 3502 Cold Tolerance

doi: 10.1128/AEM.03173-13

Figure Lengend Snippet: Strains and plasmids used in this study

Article Snippet: ATCC 3502 cbo2847 ::intron 509|510AS , Insertional disruption of cbo2847 at base 509 in antisense orientation, erm , This study.

Techniques: Plasmid Preparation, Conjugation Assay, Recombinant, Expressing

Journal: Applied and Environmental Microbiology

Article Title: The Cold-Induced Two-Component System CBO0366/CBO0365 Regulates Metabolic Pathways with Novel Roles in Group I Clostridium botulinum ATCC 3502 Cold Tolerance

doi: 10.1128/AEM.03173-13

Figure Lengend Snippet: Oligonucleotide primers used in this study

Article Snippet: ATCC 3502 cbo2847 ::intron 509|510AS , Insertional disruption of cbo2847 at base 509 in antisense orientation, erm , This study.

Techniques: Modification, Sequencing, Clone Assay, Over Expression

Journal: Applied and Environmental Microbiology

Article Title: The Cold-Induced Two-Component System CBO0366/CBO0365 Regulates Metabolic Pathways with Novel Roles in Group I Clostridium botulinum ATCC 3502 Cold Tolerance

doi: 10.1128/AEM.03173-13

Figure Lengend Snippet: Confirmation of DNA microarray results with quantitative reverse transcription-PCR (RT-qPCR). Correlation of log2 fold changes in expression of cbo0751, cbo0753, cbo1407, cbo2226, cbo2227, cbo2525, cbo2847, cbo3199, and cbo3202 between C. botulinum ATCC 3502 cbo0365 mutant and the wild-type strain 1 h after a temperature downshift from 37 to 15°C observed in the DNA microarray (x axis) and RT-qPCR (y axis) experiments.

Article Snippet: ATCC 3502 cbo2847 ::intron 509|510AS , Insertional disruption of cbo2847 at base 509 in antisense orientation, erm , This study.

Techniques: Microarray, Quantitative RT-PCR, Expressing, Mutagenesis

Journal: Applied and Environmental Microbiology

Article Title: The Cold-Induced Two-Component System CBO0366/CBO0365 Regulates Metabolic Pathways with Novel Roles in Group I Clostridium botulinum ATCC 3502 Cold Tolerance

doi: 10.1128/AEM.03173-13

Figure Lengend Snippet: Mutants of several genes under putative regulation of CBO0365 and within related metabolic pathways show impaired growth at low temperature. (A to D) Average growth of C. botulinum ATCC 3502 wild type (WT) and mutants with insertionally inactivated cbo3199 (bcd) and cbo3202 (crt) encoding two central enzymes of the ABE fermentation pathway (A), cbo1407 (bdh) and cbo2847 (ctfA) encoding components of the ABE pathway (B), arsenical resistance operon components cbo0751 (arsC) and cbo0753 (arsR) (C), and cbo2525 (phoT) encoding a phosphate ABC transporter (D) at 17°C. Error bars denote the minimum and maximum values of five biological replicates.

Article Snippet: ATCC 3502 cbo2847 ::intron 509|510AS , Insertional disruption of cbo2847 at base 509 in antisense orientation, erm , This study.

Techniques:

Journal: Genome Research

Article Title: Cancer translocations in human cells induced by zinc finger and TALE nucleases

doi: 10.1101/gr.147314.112

Figure Lengend Snippet: Induction of t(2;5)(p23;q35) translocations with TALENs. ( A ) ALCL translocations have breakpoints within the NPM1 and ALK genes on chromosomes 2 and 5, respectively, creating an NPM1–ALK fusion gene on der(5). To induce t(2;5)(p23;q35), TALENs are expressed to create DSBs (scissors) in both genes. FISH demonstrates the t(2;5)(p23;q35) translocation after TALEN expression in RPE-1 cells. Red and green signals are from an ALK probe that “breaks apart” upon translocation. The blue signal is from an NPM1 probe. Of 70 metaphases screened, two exhibited translocations and three showed breaks with the ALK break-apart probe, likely due to remaining TALEN expression at this time. ( B ) TAL NPM and TAL ALK cleavage within NPM1 and ALK introns, respectively, relevant to the NPM1–ALK translocation. DNA binding domains of TALENs are designed to bind the shaded sequences. (Arrows) DSB sites with different tail lengths representing the efficiency of cleavage in vitro, as assayed by in vitro expression of the TALENs (see Supplemental Fig. S6). TALEN cleavage activity in Jurkat cells is monitored by the T7-endonuclease assay, as shown below the sequences. ( C ) Nested PCR to detect derivative chromosomes der(2) and der(5) in Jurkat cells. Translocation breakpoint junctions are only detected after expression of both TAL NPM and TAL ALK . ( D ) RT-PCR detection of the NPM1–ALK fusion transcript after TAL NPM and TAL ALK expression in Jurkat cells and in ALCL cell line SUP-M2. The forward primer is within exon 2 of NPM1 , and the reverse primer is within exon 29 of ALK , amplifying most of the NPM1-ALK coding sequence. ( E ) Single-round PCR to detect derivative chromosomes der(2) and der(5) in Jurkat cells. Translocation breakpoint junctions are detected after expression of both TAL NPM and TAL ALK by PCR of the fragment marked (*) in C on serial dilutions of genomic DNA (50, 25, 12.5, 6.25, 3.125, and 1.56 ng). The number of times the PCR was positive for each dilution from six total experiments is indicated. The markers for 933 and 951 bp correspond to der(2) and der(5) junctions, respectively, without end modification. The larger and smaller fragments seen in some of the lanes likely correspond to junctions with large insertions or deletions. ( F ) Detection of the NPM1–ALK fusion protein in Jurkat cells after TAL NPM and TAL ALK coexpression and in the ALCL cell line SUPM2. The signal from 1 μg of cell extract from SUPM2 cells was compared with 40 μg from Jurkat cells. ( G ) Expanding pools of RPE-1 cells treated with TALENs. The PCR product corresponding to der(5) was detected when cells were split 1 to 10 every 3 d. The number of passages at each time point is indicated below the gel.

Article Snippet: Jurkat and SUDHL-1 and SUPM2 (DSMZ, Germany) cells were cultivated in RPMI-1640 medium supplemented with 10% heat inactivated FBS.

Techniques: TALENs, Translocation Assay, Expressing, Binding Assay, In Vitro, Activity Assay, Nested PCR, Reverse Transcription Polymerase Chain Reaction, Sequencing, Modification

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: 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. , 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: USO1 (13,509–1-AP) antibody to detect mouse USO1 was purchased from Proteintech.

Techniques: Gene Expression, Translocation Assay, Expressing, Quantitative RT-PCR, Control, Western Blot, Transduction, ChIP-sequencing, Binding Assay, Chromatin Immunoprecipitation, ChIP-qPCR, Quantitation Assay

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: Depletion of USO1 leads to decreased cell growth, cell cycle arrest and increased apoptosis. ( A ) Schematic representation of the pLKO5.sgRNA.EFS.tRFP lentiviral vector. Abbreviations, hU6, human U6 promoter; sgRNA, short guide RNA; sg scaf, sgRNA scaffold; tRFP, turbo red fluorescent protein. ( B ) Sample FACS plots of SEM cells transduced sequentially with Cas9 vector and sgRNA containing vector. Left, non-transduced SEM cells; middle, transduced with pLentiCas9-GFP; right, cells transduced with both pLenti-Cas9-GFP and pLKO5 vector containing USO1 -targeting sgRNA. ( C ) Western blot for USO1 in SEM cells following CRISPR/Cas9-mediated disruption of the USO1 gene using three different sgRNAs (sg1-3) and NT, non-targeting sgRNAs. ( D ) RT-qPCR measurement of USO1 in control (NT) and USO1 (sg2 & sg3) SEM cells (t test; ** P < 0.01; **** P < 0.0001) ( E ) MTS assay to study the cell growth of USO1-depleted cells (sg2 & sg3), measured as Absorbance at 490 nM (t test; ** P < 0.01; *** P < 0.001). ( F , G ) Cell cycle analysis using propidium iodide (PI) staining of control cells and USO1-depleted cells ( F ) and quantitation of cells from cell cycle analysis (Two-way Annova with Bonferroni correction; * P < 0.05; **** P < 0.0001). ( H , I ) FACS plots of Annexin V positivity in control versus USO1-depleted cells ( H ), Quantitation of cells with Annexin V positivity (t test; ** P < 0.01) ( I ).

Article Snippet: USO1 (13,509–1-AP) antibody to detect mouse USO1 was purchased from Proteintech.

Techniques: Plasmid Preparation, Transduction, Western Blot, CRISPR, Disruption, Quantitative RT-PCR, Control, MTS Assay, Cell Cycle Assay, Staining, Quantitation Assay

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: USO1 depletion significantly affects gene expression and pathways related to cell survival and proliferation. ( A ) Enrichment plots generated by the Metascape gene list enrichment analysis webtool on for the RNA-seq data from USO1 -depleted versus NT control SEM cells. The top and bottom panels show pathways that are downregulated and upregulated in USO1 -depleted SEM cells, respectively. ( B ) Volcano plot representing differentially expressed genes ( P adj < 0.01 and Log2FC > 1.5) with several examples highlighted from the pathways enriched in ( A ). ( C , D ) RT-qPCR validation of differentially expressed genes identified in ( B ) (t test; * P < 0.05; ** P < 0.01) ( E ) Western blot showing mildly reduced expression of p-MTOR (Ser2481) expression in USO1-depleted cells, compared to NT control, while MTOR remains unchanged. ( F ) Venn diagrams showing the number of shared genes between USO1-positively correlated genes in Target-Phase II ALL dataset with the genes that are significantly upregulated (top) or downregulated (bottom) in USO1 -depleted SEM cells. A hypergeometric test was utilized to compare the overlaps between the datasets using a genome size of 24,278 genes. Total and shared number of genes are indicated.

Article Snippet: USO1 (13,509–1-AP) antibody to detect mouse USO1 was purchased from Proteintech.

Techniques: Gene Expression, Generated, RNA Sequencing, Control, Quantitative RT-PCR, Biomarker Discovery, Western Blot, Expressing

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: USO1 depletion in transformed bone marrow cells shows reduced proliferation and colony forming potential. ( A ) Schematic of an in vitro model system to transform Lin - bone marrow cells from Cas9-egfp mice using overexpression of MLL-Af4 transgene in. ( B ) Analysis of overexpression of MLL-Af4 by RT-qPCR and western blot in retrovirally transduced Lin - Cas9 MLL-Af4 cells, Lin - Cas9 cells were used as negative control and 70Z/3 cells transduced with MLL-Af4 were used as positive control. RT qPCR was performed with an optimized set of primers, normalized to L32, and represented as fold-change from an internal control for each experiment. Western Blotting was performed with an antibody to MLL1. Vinculin was used as a high molecular weight loading control. ( C ) Upper panel, schematic of murine Uso1 depletion experiments, showing location of sgRNAs relative to the gene, and RT-qPCR primer location. Bottom panel, western blot analysis of 70Z/3 cells transduced with three different sgRNAs targeting Uso1 (msg1-3) cloned in the MSCV.sgRNA.mCherry.v1 vector. ( D ) RT-qPCR analysis of Uso1 depletion in 70Z/3 cells. ( E ) MTS assay (Absorbance at 490 nm) to measure proliferation in Uso1 -depleted 70Z/3 cells compared to NT control cells. ( F ) FACS plot showing gating schema to sort the Lin - Cas9 MLL-Af4 GFP + mCherry + population following transduction with the Uso1 msg3 vector. ( G ) Western blot was used to confirm the reduction in USO1 expression in sorted Lin - Cas9 MLL-Af4 cells. ( H ). Colony formation assay using Lin - Cas9 MLL-Af4 cells in methylcellulose assay as described in methods with titration of input cell number (t test, *** P < 0.001; **** P < 0.0001).

Article Snippet: USO1 (13,509–1-AP) antibody to detect mouse USO1 was purchased from Proteintech.

Techniques: Transformation Assay, In Vitro, Over Expression, Quantitative RT-PCR, Western Blot, Negative Control, Transduction, Positive Control, Control, High Molecular Weight, Clone Assay, Plasmid Preparation, MTS Assay, Expressing, Colony Assay, Methylcellulose Assay, Titration

Journal: bioRxiv

Article Title: Unbiased genome-scale identification of cis -regulatory modules in the human genome by GRAMc

doi: 10.1101/468405

Figure Lengend Snippet: To bring an insert and an N25 barcode in each reporter construct close to each other, the SCP-GFP cassette or the vector backbone is deleted by PCR with P2/nP3 or P1/P4 primers, respectively. Subsequent self-ligations of PCR products generate 2 sublibraries with N25s mated to either the 5’-end of inserts (Hs800_14) or the 3’-end of inserts (Hs800_23). Exonuclease treatment ensures survival of only mated circular ligates during subsequent second round amplification of insert::N25 cassettes with the alternate set of primers (P1/P4 for Hs800_23 and P2/nP3 for Hs800_14) to generate 2 sequencing libraries, Hs800_2314 and Hs800_1423. PCR adds PE1 and PE2 sites for Illumina paired-end sequencing. Addition of PE1 sites are accomplished using 7-phased primers per sequencing library to offset the lack of diversity in flanking adapter sequences. Phased primers incorporate 0N, 2N, 4N, 6N, 8N, 10N, and 12N random sequences between PE1 sites and respective nP3 or P4 sites. The 14 phased libraries are sequenced on the Illumina NextSeq500 platform.

Article Snippet: Sequencing libraries were prepared both for IonTorrent Proton sequencing (Batch 1: NJ197 and NJ-523; Batch2: NJ-198 and NJ-523) and Illumina NextSeq500 sequencing (14 phased libraries using NJ-400/NJ-504/NJ-505/NJ-506/NJ-507/NJ-508/NJ-509 with NJ364 or NJ-402/NJ-498/NJ-499/NJ-500/NJ-501/NJ-502/NJ-503 with NJ-399) .

Techniques: Construct, Plasmid Preparation, Amplification, Sequencing

Journal: bioRxiv

Article Title: Loss-of-function cancer-associated mutations in the EIF4G2 non-canonical translation initiation factor

doi: 10.1101/2023.08.22.554280

Figure Lengend Snippet: A HEK293T EIF4G2 KO cells were co-transfected with the indicated EIF4G2 variants and an F-LUC reporter driven by the BCL2 IRES and R-LUC reporter as an internal control. A schematic of the F-LUC reporter is shown, including a mutant A-cap structure and a hairpin upstream of the IRES sequence. F-LUC activity was quantified and normalized to the R-LUC activity; the graph shows the relative normalized LUC activity in all EIF4G2 transfectants with WT EIF4G2 transfection set as 1 (dashed red line). Total cell lysates were subjected to western blot analysis using EIF4G2 and GAPDH antibodies as loading control, shown below the graph. B,C HEK293T EIF4G2KO cells were co-transfected with the indicated EIF4G2 variants and reporters containing ROCK1 5’UTR ( B ) or WNK1 5’UTR ( C ) upstream of R-LUC along with F-LUC as internal control. Schematics of the R-LUC reporters are shown. R-LUC activity was quantified and normalized to the F-LUC activity; the graph shows the relative normalized LUC activity in all EIF4G2 transfectants with WT EIF4G2 transfection set as 1 (dashed red line). Total cell lysates were subjected to western blot analysis using EIF4G2 and tubulin or GAPDH antibodies as loading controls, shown below the graphs. Data Information: For all panels, data is presented as individual data-points and also as the mean values±S.E.M of three ( A,C ) or four ( B ) independent experiments, with a representative western blot from one of the experiments shown. Significance was determined by matched one-way ANOVA followed by Dunnett’s multiple comparison ad hoc test (comparing all variants to the WT EIF4G2 construct). Non-significant results were not indicated in the figure. A-C Schemes were created with BioRender.com.

Article Snippet: Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes, which were incubated with the indicated antibodies: mouse anti-EIF4G2 (BD Biosciences, cat# 610742, RRID:AB_398065, 1:1000 dilution), mouse anti-FLAG (Sigma-Aldrich, cat# F3165, RRID:AB_259529, 1:1000), mouse anti-GAPDH (Millipore, cat# MAB374, RRID:AB_2107445, 1:3000), rabbit anti-ROCK1 (Cell Signaling Technology, cat# 4035, RRID:AB_2238679, 1:500), rabbit anti-WNK1 (Cell Signaling Technology, cat# 4979, RRID:AB_2216752, 1:500) and mouse anti-Tubulin (Sigma-Aldrich, cat# T9026, RRID:AB_477593, 1:70000) add BCL2 (Santa Cruz Biotechnology Cat# sc-509, RRID:AB_626733, 1:1000).

Techniques: Transfection, Control, Mutagenesis, Sequencing, Activity Assay, Western Blot, Comparison, Construct