20 research outputs found

    Effects of proteasome inhibitors MG132, epoxomicin and carfilzomib on OATP1B3-mediated transport and total ubiquitin-conjugated proteins in HEK293 stable cell lines.

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    <p>HEK293-OATP1B3 cells were seeded at a density of 1.2 x 10<sup>5</sup> cells/well in 24-well plates and cultured to confluence. (A) Model-estimated fold change and associated SE in [<sup>3</sup>H]CCK-8 accumulation (1 μM, 3 min) in HEK293-OATP1B3 cells pretreated with MG132 (10 μM), epoxomicin (50 nM) and carfilzomib (200 nM) for 2 h vs. vehicle control (CTL) pretreatment. Fold changes and SE were estimated by linear mixed effects models, as described in the “Data Analysis” section (n = 3 in triplicate). To account for multiple comparisons, p-values were adjusted based on the Bonferroni method. * indicates a statistically significant difference (adjusted p<0.05) vs. CTL. (B) Immunoblot of ubiquitin was conducted in whole cell lysates of HEK293-OATP1B3 cells pretreated with MG132 (10 μM), epoxomicin (50 nM) and carfilzomib (200 nM) or vehicle control for two hours. β-actin served as the loading control for the whole cell lysates.</p

    Co-immunoprecipitation of FLAG-OATP1B1/OATP1B3 with HA-Ub.

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    <p>pCMV6-FLAG-OATP1B1 (A) or pCMV6-FLAG-OATP1B3 (B) vectors were transfected into HEK293 cells, either alone or in combination with HA-Ub expression vector. Transfection with empty vector pCMV6 served as the vehicle control in all experiments. Whole cell lysates (WCL) were subjected to immunoprecipitation (IP) with HA or FLAG antibody, followed by immunoblotting (IB) with FLAG or HA antibody, respectively. After stripping, the same blot was re-probed with HA or FLAG antibody, as indicated in the figure. Immunoblots of HA and FLAG were also conducted using WCL with β-actin serving as the loading control. Representative immunoblot images from n = 3 independent experiments are shown.</p

    Effects of bortezomib treatment on the transport kinetics of CCK-8 and surface protein levels of OATP1B3 in HEK293-OATP1B3 cells.

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    <p>HEK293-OATP1B3 cells were seeded at a density of 1.2 x 10<sup>5</sup> cells/well in 24-well plates, and were cultured for 48 h prior to performing the experiment. (A) The concentration-dependent accumulation of CCK-8 (0.1–40 μM, 3 min) was determined in HEK293-OATP1B3 cells pretreated with control (CTL) or bortezomib (Btz) (50 nM, 7 h). Solid and dashed lines represent the best fit lines of the Michaelis–Menten equation to the data of vehicle control (CTL) (closed circles) and bortezomib (Btz) pretreatment (open circles), respectively. A representative graph of three independent experiments performed in triplicate is shown. The student’s t-test was conducted to compare the V<sub>max</sub> and K<sub>m</sub> values between bortezomib and vehicle control pretreatment. * indicates a statistically significant difference (<i>p</i><0.05; bortezomib vs. CTL). (B) HEK293-OATP1B3 cells were pretreated with 50 nM bortezomib (Btz) or vehicle control (CTL) for 7 h. Surface levels of OATP1B3 were determined via biotinylation, followed by immunoblotting with OATP1B3 and Na-K-ATPase antibodies. GAPDH was used as a cytoplasmic protein marker. OATP1B3 surface protein levels were determined by densitometry and were normalized to those of Na-K-ATPase. Fold changes in the surface levels of OATP1B3 (bortezomib vs. CTL) were expressed as mean ± SD of three independent experiments.</p

    Effects of lysosome inhibitor chloroquine on total protein levels of OATP1B3 in HEK293-OATP1B3 cells and human SCH.

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    <p>HEK293-OATP1B3 cells were seeded in 24-well plates at a density of 1.2 x 10<sup>5</sup> cells/well and were cultured for 48 h prior to treatment. Human SCH were cultured as described in the Materials and Methods section. Cells were pretreated with chloroquine (CQ) (25 μM, 5 h) or vehicle control (CTL) prior to immunoblotting for OATP1B3 in HEK293-OATP1B3 cells (A) and in day 1 human SCH (B). β-actin served as the loading control in both A and B. OATP1B3 protein levels determined by densitometry were normalized to levels of β-actin. Fold changes of total protein levels of OATP1B3 in chloroquine (CQ) -treated cells vs. vehicle control treatment (CTL) were expressed as mean ± SD (n = 3).</p

    Effects of bortezomib on total OATP1B1 and OATP1 B3 protein levels and total ubiquitin-conjugated proteins in human SCH.

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    <p>Human SCH were cultured as described in the Materials and Methods section. (A) Immunoblot of OATP1B3 and OATP1B1 in whole cell lysates of human SCH that were treated with bortezomib (Btz) (50 and 250 nM) or vehicle control (CTL). β-actin served as the loading control. Representative images are shown from n = 3 and 4 donors for 50 and 250 nM treatment, respectively. (B) Fold changes of OATP1B1 and OATP1B3 protein levels. Densitometry of OATP1B1 and OATP1B3 protein levels was normalized to that of β-actin. Fold changes of total protein levels of OATP1B1 and OATP1B3 in bortezomib-treated cells vs. CTL were expressed as mean ± SD n = 3 and 4 donors for 50 and 250 nM treatment, respectively. (C) Immunoblot of ubiquitin in whole cell lysates of human SCH treated with bortezomib (Btz) (50 nM, 7 h) or vehicle control. β-actin served as the loading control. Representative images are shown from n = 3 donors.</p

    Effects of bortezomib on OATP1B3-mediated transport.

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    <p>(A) Model-estimated fold change and associated SE of [<sup>3</sup>H]CCK-8 accumulation (1 μM, 3 min) in the presence of 10–250 nM bortezomib (Btz) or 25 μM rifampicin (Rif) vs. control (CTL) in HEK293-OATP1B3 cells without any pre-incubation (Co-incubation). (B) Model-estimated fold change and associated SE of [<sup>3</sup>H]CCK-8 accumulation (1 μM, 3 min) in HEK293-OATP1B3 cells pretreated with bortezomib (Btz) vs. vehicle CTL at each indicated pretreatment concentration and time (Pre-incubation). After pretreatment, cells were washed three times with the HBSS buffer, and the [<sup>3</sup>H]CCK-8 accumulation was determined in the absence of bortezomib. (C) Model-estimated fold change and associated SE of [<sup>3</sup>H]CCK-8 accumulation (1 μM, 3 min) in human SCH pretreated with bortezomib (Btz) (50 and 250 nM, 7 h) vs. vehicle CTL. After pretreatment, cells were washed three times with the HBSS buffer, and the [<sup>3</sup>H]CCK-8 accumulation was determined in the absence of bortezomib. Model-estimated fold change and associated SE of OATP1B3-mediated [<sup>3</sup>H]pitavastatin (1 μM, 1 min) (D) and [<sup>3</sup>H]E<sub>2</sub>17βG accumulation (1 μM, 2 min) (E) in bortezomib pretreatment vs. vehicle CTL at each indicated pretreatment concentration and time (Pre-incubation). In D and E, HEK293-OATP1B3 and HEK293-Mock cells were pretreated with vehicle control (CTL) or bortezomib at the indicated concentrations and time. After washing with the HBSS buffer, OATP1B3-mediated [<sup>3</sup>H]pitavastatin (D) and [<sup>3</sup>H]E<sub>2</sub>17βG accumulation (E) was determined by subtracting the values determined in the HEK293-Mock cells from those in HEK293-OATP1B3 cells. (F) Model-estimated fold change and associated SE in [<sup>3</sup>H]CCK-8 accumulation (1 μM, 3 min) vs. CTL. Cells were pre-incubated with bortezomib-free (CTL) or 50 nM bortezomib-containing media for 2 h. At the end of pre-incubation, the culture medium was removed. After washing, CTL- and bortezomib-pretreated cells were cultured in bortezomib-free medium for the indicated time duration. [<sup>3</sup>H]CCK-8 (1 μM, 3 min) accumulation was determined at the indicated time points after washing three times (<i>n</i> = 3 in triplicate). A generalized linear mixed model was fit to the data in A-F as described in the “Materials and Methods” (n = 3 for A, D-F; n = 6 for B; n = 5 for C; all experiments were performed in triplicate). To account for multiple comparisons, p-values were adjusted based on the Bonferroni method. * indicates a statistically significant difference (adjusted <i>p</i><0.05) vs. CTL.</p

    Disposable Copper-Based Electrochemical Sensor for Anodic Stripping Voltammetry

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    In this work, we report the first copper-based point-of-care sensor for electrochemical measurements demonstrated by zinc determination in blood serum. Heavy metals require careful monitoring, yet current methods are too complex for a point-of-care system. Electrochemistry offers a simple approach to metal detection on the microscale, but traditional carbon, gold (Au), or platinum (Pt) electrodes are difficult or expensive to microfabricate, preventing widespread use. Our sensor features a new low-cost electrode material, copper, which offers simple fabrication and compatibility with microfabrication and PCB processing, while maintaining competitive performance in electrochemical detection. Anodic stripping voltammetry of zinc using our new copper-based sensors exhibited a 140 nM (9.0 ppb) limit of detection (calculated) and sensitivity greater than 1 μA/μM in the acetate buffer. The sensor was also able to determine zinc in a bovine serum extract, and the results were verified with independent sensor measurements. These results demonstrate the advantageous qualities of this lab-on-a-chip electrochemical sensor for clinical applications, which include a small sample volume (μL scale), reduced cost, short response time, and high accuracy at low concentrations of analyte

    TCF/Lef factors up-regulate UCH L1 expression through direct binding to its promoter.

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    <p>A. TCF4 binds to endogenous uch l1 promoter. ChIP/PCR analysis was performed to determine binding of TCF4 factor to two putative binding sites on uch l1 promoter (left) with the use of specific TCF4 antibody in KR4 or 293 cells treated with 25 mM LiCl. Normal IgGs were used as a negative controls in IPs. PCR reactions were performed with primers targeting TCF4 binding sites on the uch l1 promoter (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005955#s3" target="_blank">Materials and Methods</a>), and amplified DNA products were resolved in 2% agarose gel. B. Mutations in TCF/Lef putative sites inhibit β-catenin/TCF4-dependent activation of the uch l1 promoter. Site-directed mutagenesis of TCF/Lef binding sites was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005955#s3" target="_blank">Materials and Methods</a>. 3T3 cells were transfected with Uchl1p-Luc wild type and mutant reporter plasmids in the presence or absence of TCF4 expression vector. Luciferase assays were performed 48 h post-transfection and are normalized to β-gal activity. Where indicated 25 mM LiCl was added to the cells 6 h before harvesting. Expression of TCF4 was confirmed by Western blotting.</p

    New isocoumarin and stilbenoid derivatives from the tubers of <i>Sparganium stoloniferum</i> (Buch.-Ham.)

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    <p>A new isocoumarin derivative 8,5′-dihydroxy-6′-methoxy-4-phenyl-5,2′-oxidoisocoumarin (<b>1</b>) and a new stilbenoid derivative methyl 5-hydroxy-2-(2-hydroxyphenyl)benzofuran-4-carboxylate (<b>2</b>) together with nine known compounds (<b>3</b>–<b>11</b>) were isolated from the tubers of <i>Sparganium stoloniferum</i> Buch.-Ham.. Another stilbenoid derivative (<b>3)</b> and a xanthone (<b>4)</b> were identified as new natural products and compounds <b>5</b>–<b>10</b> were obtained for the first time from the genus <i>Sparganium</i>. All their structures were elucidated by comprehensive spectroscopic analysis and comparison with available literature information.</p
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