15 research outputs found

    Representative confocal fluorescence micrographs of NRVMs grown on cover glasses.

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    <p>Ad-GFPu infection and BFA (10 nM) treatment are as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100715#pone-0100715-g004" target="_blank"><b>Figure 4</b></a>. At 24 hr after BFA treatment, the cells were fixed with 4% paraformaldehyde and subject to immunofluorescence staining for p62 (red). GFPu direct fluorescence (green) and p62 indirect immunofluorescence were visualized and captured using a confocal microscope. In the BFA treated cells, both p62 and GFPu are increased and the increased GFPu is co-localized with p62. Scale bar = 50 µm.</p

    Inhibition of the ALP stabilizes a UPS substrate.

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    <p>NRVMs were cultured and infected with Ad-GFPu/Ad-RFP viral mixture as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100715#pone-0100715-g004" target="_blank"><b>Figure 4</b></a>. Forty-eight hours later, the cells were treated with 3-MA (2 mM, <b>A</b> and <b>B</b>) or BFA (10 nM, <b>C</b> and <b>D</b>), or respective vehicle control (CTL) for 12 hrs; then, the treatment of cycloheximide (CHX, 50 µM) was initiated. Immediately before (0 min) or 5, 10, 15, 30, 60, and 120 min after CHX administration, the cells were harvested for western blot analyses for GFPu and RFP. Representative western blot analysis images (<b>A</b>, <b>C</b>) and densitometry data pooled from 3 biological repeats (<b>B</b>, <b>D</b>) are shown. *<i>p</i><0.05, **<i>p</i><0.01 vs. CTL.</p

    p53 protein binds to the <i>RASSF1A</i> promoter.

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    <p><b>A</b>, EMSA analysis of p53 binding to the <i>RASSF1A</i> promoter. WT, the wild type p53 binding site probe from human <i>RASSF1A</i> promoter; Mut, mutant p53 binding site probe; ds, two complementary oligonucleotide probes; ss, single stranded probe. The supershift band in lane 2 contains the anti-p53/wt DNA/p53 protein complex. Complexes were separated by native gel electrophoresis. Mutant sequence was showed in lower panel. <b>B</b>, ChIP assay of p53 binding to the <i>RASSF1A</i> promoter in Siha cells. Samples of sonicated chromatin from Siha cells cross-linked in 1% formaldehyde were immunoprecipitated with anti-p53 antibody, preimmuno IgG (IgG) and no antibody (beads only). DNA isolated from immunoprecipitated material was amplified by PCR with primers to amplify the 147 bp <i>RASSF1A</i> promoter sequences flanking the p53 binding site (−2799 bp to −2653 bp). A region of 192 bp flanking intron 1 and exon 2 of the <i>RASSF1A</i> was amplified as a control. Input lanes represent sonicated chromatin samples as positive control. The amplified PCR fragments were analysed on 2% agarose gel.</p

    Lysosomal inhibition does not alter proteasome abundance and proteasome peptidase activities in mouse hearts.

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    <p>Mice were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100715#pone-0100715-g001" target="_blank">Figure 1</a>. <b>A</b>, Representative images of western blot analyses for the representative subunits of the 19S proteasome (Rpt6) and the 20S proteasome (α3, α4, β2, and β5) in the total protein extracts from ventricular myocardium. <b>B</b>, Pooled densitometry data of β2 and β5 subunits as analyzed in A. <b>C</b>, Proteasome peptidase activity assays. Synthetic fluorogenic substrates specific for chyomtrypin-like, caspase-like, and trypin-like peptidases were used to measure the proteasomal peptidase activities in the crude protein extracts from ventricular myocardium collected from mice treated with BFA or vehicle control (CTL) for 24 hours. The assay was performed in the absence (-) or presence (+) of ATP to detect the activity of the 20S and the 26S proteasome, respectively. Proteasome inhibitor-suppressible activities were attributed to the proteasome. N = 4 mice/group. **<i>p</i><0.01 vs. the respective group without ATP.</p

    Immunohistochemical staining of RASSF1A in human testis cancer.

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    <p>Normal testis sample (<b>A</b>) and testis tumor with methylated RASSF1A (<b>B</b>). Normal testis section stained positively for RASSF1A. RASSF1A was mainly expressed in cytoplasm of the spermatogonia (SG), spermatocytes (SC), Sertoli cells (Sn) and Leydig cells (LC) of normal testis, while seminoma sample with methylated RASSF1A showed weak staining. Nuclei were stained with Hematoxlin.</p

    Promoter activity and methylation analysis of <i>RASSF1A</i> in human testis cancer.

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    <p><b>A</b>, <i>RASSF1A</i> promoter activity by GFP expression under the control of different length of RASSF1A promoter segments. A predicted p53 binding site in <i>RASSF1A</i> promoter (−2718 bp) was showed in the construction c. <b>B</b>, CpG island prediction of <i>RASSF1A</i> by software MethPrimer online (<a href="http://www.urogene.org/methprimer/" target="_blank">http://www.urogene.org/methprimer/</a>). Predicted CpG island is indicated by blue color. <b>C</b>, Representative results of the methylation-sensitive PCR (MSP) analysis of <i>RASSF1A</i> in both seminoma and nonseminoma. M, MSP PCR; U, unmethylation-sensitive PCR. <b>D</b>, DNA methylation status of individual CpG sites by sodium bisulfite sequencing analysis. Black and white circles represent methylated and unmethylated CpGs respectively.</p

    Dynamic effects of ALP inhibition via 3-MA or BFA on p62 protein levels and a surrogate UPS substrate in cultured neonatal rat ventricular myocytes (NRVMs).

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    <p>Cultured NRVMs were infected with adenoviruses to express GFPu and RFP (Ad-GFPu, Ad-RFP) 48 hr before 3-methyladenine (3-MA) or bafilomycin A1 (BFA) treatment. <b>A ∼ C</b>, The time course of protein level changes in the GFPu/RFP ratio and the p62/β-tubulin ratio in NRVMs treated with 3-MA (3 mM) or vehicle control. Representative images of western blots (<b>A</b>) and quantitative data from 3 repeats (<b>B, C</b>) are shown. Paired <i>t</i>-test, *<i>p</i><0.01. <b>D</b> and <b>E</b>, 3-MA and BFA concentration-dependently increases GFPu/RFP ratio at 24 hours. At 24 hr after the drug treatment, the cells were harvested for total protein extraction and western blot analyses of the indicated proteins. Representative images (<b>D</b>) and pooled densitometry data (<b>E</b>) are shown. Two-way ANOVA followed by the Scheffé's test; *<i>p</i><0.05, **<i>p</i><0.01 vs. CTL; n = 3 repeats/group.</p

    Lysosomal inhibition accumulates both autophagic and proteasomal substrates in mice: hearts and kidneys.

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    <p>Male GFPdgn transgenic mice at 10 weeks of age were treated with bortezomib (BZM, 1 mg/kg, i.p.), bafilomycin A1 (BFA, 2.5 mg/kg/12 hrs, i.p.), or vehicle control (CTL). The mice were sacrificed at either 3 or 24 hours after the first injection and myocardial and kidney tissues were collected for total protein and RNA extraction and subsequent analysis. <b>A</b> and <b>B</b>, Representative images (A) and pooled of densitometry data (B) of western blot analyses for myocardial GFPdgn and other indicated proteins. <b>C</b>, Representative images (upper) and pooled densitometry data (lower) of western blot analyses for renal GFPdgn protein levels. <b>D</b> and <b>E</b>, Representative images (upper) and pooled densitometry data of reverse transcription (RT) PCR analyses of GFPdgn mRNA levels in heart and kidney tissues. Total RNA was used for the RT to synthesize the first strand cDNA which was subsequently used for GFPdgn and GAPDH duplex RT-PCR. GAPDH was probed as loading control. N = 4 mice/group. *<i>p</i><0.05, **<i>p</i><0.01 vs. CTL.</p

    Down-regulation of <i>RASSF1A</i> by p53.

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    <p><b>A</b>, <i>RASSF1A</i> promoter activity was inhibited by p53 protein with a dose dependent manner. pRASSF1A-GFP (construction c in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017017#pone-0017017-g001" target="_blank">Fig. 1</a>) was co-transfected with different concentration of CMV-p53 into COS-7 cells, and transfected cells were analyzed by the flow cytometry. <b>B</b>, pRASSF1A-GFP was transfected into COS-7 cells (<b>a</b>) or co-transfected with p53 (<b>b</b>), and images were taken under fluorescent microscopy. p53 remarkably decreased expression of RASSF1A-GFP. GFP was mainly expressed in the cytoplasm. Nuclei were stained with Hoechst. <b>C</b>, Overexpression of p53 in Siha cells decreased RASSF1A protein levels, revealed by Western blot analysis of RASSF1A and p53 expression using anti-RASSF1A or p53 antibody after transfection of the p53 expression plasmid into Siha cells. Lysates of non-transfected cells and cells transfected with the pcDNA3 vector were used as controls. β-actin was used as an internal control. Molecular weights of the proteins are shown on the right.</p

    Autophagic inhibition accumulates GFPu in a p62 dependent manner.

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    <p>Cultured NRVMs were infected with adenoviruses to express GFPu and RFP (Ad-GFPu/RFP) 24 hours before transfection with siRNA for Atg7 (siAtg7), Rab7 (siRab7) and/or for p62 (sip62). A luciferase-specific siRNA (siLuc) was used as control for siRNA infection and off-target effects. The cells were harvested at 48 h later for extracting total proteins. <b>A</b>, Shown are representative images out of 3 repeats. <b>B</b>, pooled densitometry data. *p<0.05 vs. CTL.</p
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