10 research outputs found

    Loss of PTP expression in apoptotic cells leads to an increase in cell proliferation.

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    <p>PTP gene expression was silenced in ER-dnE2F1 cells with siRNA specific for PTP-1B (+PTP-1B), SHP-2 (+SHP-2), or PTEN (+PTEN). Uninduced and induced control cells were transfected with scrambled siRNA. Then cells were treated with 4-OHT for 48 h to initiate Rb/E2F-associated apoptosis. Uninduced control cells were left untreated. (A) Total protein was isolated from lysated cells, resolved by SDS-PAGE, and immunoblotted with antibodies specific for PTP-1B, SHP-2, PTEN and β-actin. (B) Morphological changes in uninduced and induced ER-dnE2F1 cells were compared with morphological changes in induced cells after knockdown of PTPs. (C) The number of cells within three random fields (100X) were counted and results are expressed as the mean + standard deviation. (D) Cell viability was determined by WST-8 assay in two independent experiments performed in triplicate. Results are expressed as the mean + standard deviation. (*) <i>p</i><0.05; (**) <i>p</i><0.01; (***) <i>p</i><0.001.</p

    SHP-2 and PTEN protein expression increases during Rb/E2F-associated apoptosis.

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    <p>ER-dnE2F1 cells were treated with 4-OHT for the indicated times to induce Rb/E2F-associated apoptosis. Control cells (0 h) were left untreated. (A) Total protein was isolated from lysated cells at the indicated time points following induction of apoptosis, resolved by SDS-PAGE, and immunoblotted with antibodies specific for PTP-1B, SHP-2, PTEN and β-actin. (B) Relative expression levels of PTP-1B, SHP-2, and PTEN were quantified by densitometry. (h) Hours.</p

    Knockdown of PTP expression leads to a reduction in caspase activity.

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    <p>ER-dnE2F1 cells were transfected with siRNA specific for PTP-1B (+PTP-1B), SHP-2 (+SHP-2), or PTEN (+PTEN). Uninduced and induced control cells were transfected with scrambled siRNA. Then cells were treated with 4-OHT for 24 or 48 h to initiate Rb/E2F-associated apoptosis. Uninduced control cells were left untreated. (A) Caspase-3/CPP32 cleavage was measured by colorimetric assay in three independent experiments. Results are expressed as the mean + standard error. (B) FLICE/Caspase-8 cleavage was measured by colorimetric assay in three independent experiments. Results are expressed as the mean + standard error. (C) Total protein was isolated from lysated cells at 24 h following induction of apoptosis, resolved by SDS-PAGE, and immunoblotted with antibodies specific for cleaved caspase-3 (Denoted by arrow), caspase-3 and β-actin. (*) <i>p</i><0.05; (**) <i>p</i> = 0.01; (***) <i>p</i><0.01.</p

    <i>PTEN</i> transcription is directly regulated by Rb/E2F complexes.

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    <p>ER-dnE2F1 cells were treated IPTG and with 4-OHT to induce Rb/E2F-associated apoptosis. Uninduced control cells were left untreated. (A) Total RNA was isolated at the indicated time points following induction of apoptosis and used for qPCR analysis for two independent experiments performed in triplicate. Results are expressed as the mean + standard deviation. (B) Chromatin was isolated from the cells, sheared and immunoprecipitated with control mouse IgG or E2F1 antibody. A ∼300 bp sequence within the PTEN promoter region was amplified by PCR. GAPDH was used as control. (−) Untreated; (+) Treated; (NA) No antibody negative control; (*) No chromatin negative control.</p

    Genetic modifiers in Venezuelan HD subjects.

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    <p>Mixed effect linear regression analysis was performed for each SNP to determine the level of significance of the SNP in explaining the variance in residual age at onset in Venezuelans. SNPs that have minor allele frequency greater than 5% are shown, revealing genome-wide significant signals on chromosome 7. The X-axis and Y-axis represent chromosome and -log10(p-value), respectively. A dotted red horizontal line marks the level for genome-wide significance.</p

    Sequence of hap.03 mutant haplotype.

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    <p>(A) Based on consensus alleles in 7 phased haplotypes in the sequencing family, the hap.03 disease haplotype sequence was reconstructed at the nucleotide level. Alleles on hap.03 that are different from the reference allele are shown with their genomic locations relative to <i>HTT</i> refseq NM_002111. The previously reported sequence of hap.01 is also displayed. D, I and N represent deletion, insertion, and missing, respectively. (B) The region containing the CAG repeat was missing in the sequencing data due to difficulty in aligning repeats. Thus, we performed additional Sanger sequencing to determine the sequence of the <i>HTT</i> CAG repeat region and found that the transmitting HD parent has a typical repeat sequence and downstream elements: uninterrupted 45 CAG repeats, 1 CAA, 1 CAG, 1 CCG, 1 CCA, 7 CCG, and 2 CCT, comparable to the structure seen on the most frequent HD hap.01 chromosome in Europeans.</p

    Earlier age at onset in HD subjects in Venezuela.

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    <p>(A) We evaluated the relationship between age at motor onset and the longer CAG repeat using the QC-passed data set. Age at onset and the longer CAG repeat of HD homozygotes are indicated in open red symbols (12 individuals). (B) Age at onset of motor signs was plotted against the size of expanded CAG repeat for HD subjects with European ancestry (blue) and Venezuelan HD subjects (red). Subsequently, log transformed age at onset (natural log) was modeled as a function of CAG repeat length for each population in a linear regression model. Then, fitted values were transformed back into natural scale age at onset for plotting. Blue and red lines represent regression models for European and Venezuelan HD, respectively. In addition, for a given CAG repeat size, the average of age at onset of HD subjects carrying a hap.01 disease haplotype (circle) or a hap.03 disease haplotype (triangle) are marked. (C) Disease duration data were compared between European and Venezuelan HD subjects. Duration values were available for 645 European HD and 85 Venezuelan HD subjects. Means of duration were 15.4 and 15.7 for European and Venezuelan HD subjects, respectively (Wilcoxon test p-value = 0.2783).</p

    Genome-wide significant modification signals on chromosome 7 in Venezuelan HD.

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    <p>Association analysis results in Venezuelan HD (A) and in European HD (B) are displayed for the chromosome 7 region. Each grey circle represents a SNP. Y-axis and X-axis represent significance (-log10(p-value)) and genomic coordinate (hg19 assembly), respectively. Dotted red lines mark the level for genome-wide significance. Genes on the plus strand and negative strand are displayed in red and blue, respectively. Recombination rate based on HapMap data is shown as a blue trace (secondary Y-axis). (C) For the top SNP in this region in Venezuelan HD analysis, allele frequency, effect size, and p-values are shown for both Venezuelan HD and European HD. (D) In addition, a conditional analysis was performed focusing on the chromosome 7 region in Venezuelan HD subjects to determine whether a cluster of significant SNPs in this region tags a single modification signal. SNPs in this region were conditioned by the top SNP in the association analysis. Y-axis and X-axis represent significance in the conditional analysis and original association analysis.</p

    Improvement of chromosome 15 association signals by addition of Venezuelan HD data.

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    <p>(A) GWA analysis results for European HD subjects were combined with those of Venezuelans in order to determine whether or not addition of Venezuelan data improved modification association signals. Association signals in Europeans (X-axis) and that in Europeans + Venezuelans (Y-axis) are plotted. Open grey circles, which are above the red diagonal line, represent SNPs whose significance in association analysis was improved when European and Venezuelan data were combined. (B) A regional association plot was generated focusing on chromosome 15, revealing that rs150393409, which specifies an arginine to histidine missense alteration predicted to be deleterious to FAN1, is the most significant SNP in this region. (C) A summary table is shown focusing on the top SNP in European + Venezuelan HD analysis.</p

    A potential role for reduced <i>SOSTDC1</i> expression levels in HD onset modification.

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    <p>Modifier-associated SNPs in the chromosome 7 region were checked for association with expression levels of <i>SOSTDC1</i>. The Y- and X-axes represent significances in GTEx eQTL and modification in our GWA analysis, respectively. Panel A shows SNPs whose minor alleles are associated with decreased expression levels of <i>SOSTDC1</i>. Panel B shows SNPS whose minor alleles are associated with increased expression levels of <i>SOSTDC1</i>.</p
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