Journal: Oncotarget

Article Title: Identification of Mubritinib (TAK 165) as an inhibitor of KSHV driven primary effusion lymphoma via disruption of mitochondrial OXPHOS metabolism

doi: 10.18632/oncotarget.27815

Figure Lengend Snippet: ( A ) Graph of cell cycle kinetics data comparing Mubritinib (MUB) to cytarabine (CYT) and rapamycin (RAP). DMSO and camptothecin (CPT) were used as controls. PEL cells (BC1 and BCBL1) treated with 7.5 nM Mubritinib show a significant decrease in the total population of S and G2 cells that is not observed for BJAB or LCL352 cells. Neither cytarabine nor rapamycin showed similar selectivity for inhibition of PEL cell growth (S, G2) ( ** p < 0.001, * p < 0.05; Student’s T Test). ( B ) Cell cycle profiles comparing cells (BJAB, BC1, BCBL1, and LCL352) treated with DMSO and 15 nM Mubritinib measured by FACS flow cytometry analysis of propidium iodide staining. ( C ) Flow cytometry analysis of Annexin V/PI staining comparing cells (BJAB, BC1, BCBL1, LCL352) treated with DMSO and 15 nM Mubritinib. ( D ) Graphs summarizing the decrease in live cell populations observed from the Annexin V/PI experiment. Camptothecin (4 mM) and Cytarabin (1 mM) are shown for comparison. ( ** p < 0.001; Mann–Whitney). ( E ) ChIP-qPCR assay for LANA or IgG control in BCBL1 cells treated with Mubritinib (15 nM) for 72 hrs with primers for KSHV TR, control region a, or cellular Actin. ( F ) Western blot control of ChIP assays showing LANA, RTA, Actin, or gH2AX in BCBL1 cells at 48 h after addition of DMSO (–) or Mubritinib (15 nM) (+).

Article Snippet: Adrucil (228440010) and Cytarabine (449561000) were purchased from Acros Organics.

Techniques: Inhibition, Flow Cytometry, Staining, Comparison, MANN-WHITNEY, ChIP-qPCR, Control, Western Blot

Journal: Molecular Microbiology

Article Title: The archaeal RNA polymerase subunit P and the eukaryotic polymerase subunit Rpb12 are interchangeable in vivo and in vitro

doi: 10.1111/j.1365-2958.2008.06577.x

Figure Lengend Snippet: A. ribbon model of subunit P from Sulfolobus solfataricus ( Hirata et al ., 2008 ) generated using PyMOL. In the Sulfolobus structure the corresponding structural elements of the Pyrococcus subunit analysed and mutated in this study are shown with side chains and labelled by colours. A 17-amino-acid C-terminal peptide analyzed here is shown in yellow, the N-terminus in green, the cysteine residues 27 and 30 of the zinc-ribbon motif which were replaced by S are shown in red, R26 replaced by A in blue and S32 (Y in Sulfolobus ) replaced by A in black. B. alignment of the protein sequence of P subunits from eight archaeal species and two yeast Rpb12 (Rpc10) sequences. The alignment was generated using ClustalW version 2.0 ( Larkin et al. , 2007 ). The alignment was displayed using the ESPript software ( Gouet et al ., 1999 ). The consensus sequence displayed was calculated with Risler matrix and a similarity global score of 0.75% (uppercase is identity, lowercase is consensus level). The sequence of the C-terminal peptide analyzed in cell-free transcription assays is labelled with a yellow box and the single point mutations introduced into subunit P are indicated with arrows. The numbers in the first line indicate the Pyrococcus furiosus RpoP amino acid sequence from 1 to 49. The numbers in the last line shown in italics indicate the Saccharomyces cerevisiae Rpb12 amino acid sequence from position 1 to 70.

Article Snippet: The YCplac22 vector encoding TRP1 supported growth on tryptophan-free minimal media (SDC; MP Biomedicals) and after counterselection on FOA against pFL44- RPC10 growth was monitored at four temperatures (18, 25, 30 and 37°C) by plating on minimal media containing 5-fluoroorotic acid (1 g l −1 ; Toronto Research Chemicals) as described previously ( ).

Techniques: Generated, Sequencing, Software

Journal: Molecular Microbiology

Article Title: The archaeal RNA polymerase subunit P and the eukaryotic polymerase subunit Rpb12 are interchangeable in vivo and in vitro

doi: 10.1111/j.1365-2958.2008.06577.x

Figure Lengend Snippet: Archaeal rpoP can complement the essential function of yeast RPB12. DNA regions encoding RpoP of Pyrococcus furiosus (C1) were fused with the strong yeast RPS28B promoter and inserted in the centromeric plasmid YCplac22. The resulting constructs were transformed into yeast strain YGVS019 carrying chromosomal deletion of RPB12 rescued by a RPB12 / URA3 plasmid (pFL44- RPC10 ). Transformants were transferred on 5-fluoroorotic acid containing plates to counterselect against plasmid pFL44- RPC10 and plates were incubated at 25°C for 5 days. The cells expressing RpoP (C1) showed growth comparable to cells expressing yeast RPB12 (C4) at 25°C and reduced growth at 37°C (data not shown). A similar construct with a chimeric gene encoding for the N-terminal domain of Rpb12 fused with rpoP of Pyrococcus (C3) could also complement the essential function of RPB12 and did not lead to temperature sensitive growth phenotype. The N-terminal domain coding region of RPB12 alone (C2) could not complement the essential functions of RPB12 .

Article Snippet: The YCplac22 vector encoding TRP1 supported growth on tryptophan-free minimal media (SDC; MP Biomedicals) and after counterselection on FOA against pFL44- RPC10 growth was monitored at four temperatures (18, 25, 30 and 37°C) by plating on minimal media containing 5-fluoroorotic acid (1 g l −1 ; Toronto Research Chemicals) as described previously ( ).

Techniques: Plasmid Preparation, Construct, Transformation Assay, Incubation, Expressing