Supporting data Fig 3A, 3B, 4D, S6 from Preclinical Anticancer Efficacy of BET Bromodomain Inhibitors Is Determined by the Apoptotic Response
Andrew R. ConeryRichard C. CentoreKerry L. SpillaneNicole E. FollmerArchana Bommi‐ReddyCharlie HattonBarbara M. BryantPatricia GreningerArnaud AmzallagCyril H. BenesJennifer A. MertzRobert J. Sims
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<p>This file contains the following: source data for the graphs shown in Figure 3A (GI50, Z sub G1, and Z G1 increase); source data for the heatmap and graph shown in Figure 3B (expression fold change, log2 fold change, and gene score); qPCR source data for the heatmap and graph in Figure 4D; measurements of Western blotting band intensity from Figure S6.</p>Mosaic virus
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The disruption of six novel genes (YDL059c, YDL060w, YDL063c, YDL065c, YDL070w and YDL110c), localized on the left arm of chromosome IV in Saccharomyces cerevisiae, is reported. A PCR-based strategy was used to construct disruption cassettes in which the kanMX4 dominant marker was introduced between two long flanking homology regions, homologous to the promoter and terminator sequences of the target gene (Wach et al., 1994). The disruption cassettes were used to generate homologous recombinants in two diploid strains with different genetic backgrounds (FY1679 and CEN.PK2), selecting for geneticin (G418) resistance conferred by the presence of the dominant marker kanMX4. The correctness of the cassette integration was tested by PCR. After sporulation and tetrad analysis of the heterozygous deletant diploids, geneticin-resistant haploids carrying the disrupted allele were isolated. YDL060w was shown to be an essential gene for vegetative growth. A more detailed phenotypic analysis of the non-lethal haploid deletant strains was performed, looking at cell and colony morphology, growth capability on different media at different temperatures, and ability to conjugate. Homozygous deletant diploids were also constructed and tested for sporulation. Only minor differences between parental and mutant strains were found for some deletant haploids. Copyright © 1999 John Wiley & Sons, Ltd.
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It has been shown that γ-irradiation induces apoptosis of the human promyeloid leukemia cell line HL-60, but the mechanism remains unclear. To explore the effect of caspase-3 in this apoptotic model, antisense oligodeoxynucleotides (ASODNs) targeting 5′-noncoding region (ASODN-1) and initial translation region (ASODN-2) of caspase-3 mRNA were designed, synthesized and introduced into HL-60 cells by means of liposome-mediated transfection followed by γ-irradiation in the present study. The TUNEL assay was used for morphological analysis of HL-60 cell apoptosis. Immunocytochemical staining, Western blotting and RT-PCR were, respectively,performed for detecting expression of caspase-3 and its mRNA. HL-60 cells transfected with mismatched oligodeoxynucleotide (MODN) or untransfected were taken as the control groups. The TUNEL assay showed that the percentages of HL-60 cell apoptosis induced by γ-Irradiation in both ASODN-1 and ASODN-2 groups were significantly reduced compared with those in the control group (P0.01) when the final transfection concentration was ≥3μmol/L. Immunocytochemistry demonstrated that in the ASODN-1 and ASODN-2 groups, caspase-3 positive cell percentages were reduced and average gray values of the positive cells increased significantly compared with those in the control group (P0.01). Western blotting found that procaspase-3 expression in HL-60 cells of the ASODNs groups was decreased,and it was lower in the ASODN-1 group than in the ASODN-2 group. RT-PCR revealed marked expression of caspase-3 mRNA in HL-60 cells of the control group. Expression of caspase-3 mRNA was decreased after ASODNs transfection. Furthermore, ASODN-1 was more effective in inhibiting HL-60 cell apoptosis (P0.05) and caspase-3 expression (P0.01) than ASODN-2. These results indicate that caspase-3 mRNA ASODNs prevent HL-60 cells from apoptosis induced by γ-radiation,and reduce expression of caspase-3 and its mRNA. These effects are dose dependent in a certain range.
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