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  • 95
    ATCC a375 cells
    CEP350 is a melanoma tumor suppressor. ( a ) CEP350 alterations encoded in human melanoma genomes. Amino acid positions in gray, black and red text denote synonymous, nonsynonymous and non-damaging, and nonsynonymous and damaging alterations, respectively. Single and double asterisks denote 2 genomes with ≥2 mutations. ( b–e ) CEP350 depletion accelerates xenograft progression in vivo . Primary shRNA experiment consisting of 1 million <t>A375</t> cells stably expressing control shNTC construct (MOI of 6; n = 10) or 3 non-overlapping, pooled shRNA constructs for CEP350 (clones 689, 691 and 694, each at MOI of 2; n = 10) injected subcutaneously into immunodeficient NSG mice. ( b ) Xenograft volumes calculated over a 1-month period. ( c ) The cohorts receiving the CEP350 shRNA pool grew significantly faster than matched cohorts receiving control shNTC. The growth curves differed significantly ( P
    A375 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 95/100, based on 267 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher advanced dmem
    Morphological characteristics and functional analysis of cell line–derived ESCC 3D organoids. ESCC 3D organoids were grown with TE11 cells in indicated cell culture media to acquire ( A ) phase contrast images and evaluate ( B ) organoid growth (size) at indicated time points. ( C ) Organoid formation rate was determined at day 11. RPMI1640 and <t>DMEM</t> are supplemented with 10% FBS. <t>aDMEM/F12</t> + is fully supplemented as used to grow patient-derived 3D organoids. Scale bar =100 μm. ( B ) Data are presented as mean ± SD of at least 7 organoids. Data represent 3 independent experiments with similar results and 1-way analysis of variance with multiple comparisons (Tukey) was performed for each ( B ) time point in and ( C ) condition. * P
    Advanced Dmem, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 851 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    GE Healthcare dulbeccos s modified eagle s medium dmem
    Morphological characteristics and functional analysis of cell line–derived ESCC 3D organoids. ESCC 3D organoids were grown with TE11 cells in indicated cell culture media to acquire ( A ) phase contrast images and evaluate ( B ) organoid growth (size) at indicated time points. ( C ) Organoid formation rate was determined at day 11. RPMI1640 and <t>DMEM</t> are supplemented with 10% FBS. <t>aDMEM/F12</t> + is fully supplemented as used to grow patient-derived 3D organoids. Scale bar =100 μm. ( B ) Data are presented as mean ± SD of at least 7 organoids. Data represent 3 independent experiments with similar results and 1-way analysis of variance with multiple comparisons (Tukey) was performed for each ( B ) time point in and ( C ) condition. * P
    Dulbeccos S Modified Eagle S Medium Dmem, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 78/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    82
    Lonza complete dulbeccos s modified eagle s medium dmem
    Morphological characteristics and functional analysis of cell line–derived ESCC 3D organoids. ESCC 3D organoids were grown with TE11 cells in indicated cell culture media to acquire ( A ) phase contrast images and evaluate ( B ) organoid growth (size) at indicated time points. ( C ) Organoid formation rate was determined at day 11. RPMI1640 and <t>DMEM</t> are supplemented with 10% FBS. <t>aDMEM/F12</t> + is fully supplemented as used to grow patient-derived 3D organoids. Scale bar =100 μm. ( B ) Data are presented as mean ± SD of at least 7 organoids. Data represent 3 independent experiments with similar results and 1-way analysis of variance with multiple comparisons (Tukey) was performed for each ( B ) time point in and ( C ) condition. * P
    Complete Dulbeccos S Modified Eagle S Medium Dmem, supplied by Lonza, used in various techniques. Bioz Stars score: 82/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Thermo Fisher high glucose dulbeccos s modified eagle s medium
    Morphological characteristics and functional analysis of cell line–derived ESCC 3D organoids. ESCC 3D organoids were grown with TE11 cells in indicated cell culture media to acquire ( A ) phase contrast images and evaluate ( B ) organoid growth (size) at indicated time points. ( C ) Organoid formation rate was determined at day 11. RPMI1640 and <t>DMEM</t> are supplemented with 10% FBS. <t>aDMEM/F12</t> + is fully supplemented as used to grow patient-derived 3D organoids. Scale bar =100 μm. ( B ) Data are presented as mean ± SD of at least 7 organoids. Data represent 3 independent experiments with similar results and 1-way analysis of variance with multiple comparisons (Tukey) was performed for each ( B ) time point in and ( C ) condition. * P
    High Glucose Dulbeccos S Modified Eagle S Medium, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 79/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher dmem
    Modulation of the nonenzymatic conversion of MPDP + into MPP + . (A) MPDP + (100 μM) was kept under atmospheric conditions in <t>DMEM</t> medium at <t>37°C</t> or under oxygen-free conditions (B) for the time intervals indicated. MPP + , MPDP + , and MPTP were detected by HPLC. (C, D) MPDP + (100 μM) was maintained in potassium phosphate buffer with varying pH, as indicated in each time interval. Autoxidation of MPDP + into MPP + was monitored photospectrometrically. (E, F) MPDP + (100 μM) was kept under atmospheric (E) or oxygen-free conditions (F) in potassium phosphate buffer of different pH at 37°C for the indicated incubation intervals. (G, H) To identify the influence of metabolites of the dopamine pathway and physiologically relevant iron-containing molecules, MPDP + (100 μM) was treated with the indicated compounds (100 μM each) under aerobic conditions with a pH of 7.4. All data are mean ± SD ( n = 8) * p
    Dmem, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 147960 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    AddexBio dmem
    Modulation of the nonenzymatic conversion of MPDP + into MPP + . (A) MPDP + (100 μM) was kept under atmospheric conditions in <t>DMEM</t> medium at <t>37°C</t> or under oxygen-free conditions (B) for the time intervals indicated. MPP + , MPDP + , and MPTP were detected by HPLC. (C, D) MPDP + (100 μM) was maintained in potassium phosphate buffer with varying pH, as indicated in each time interval. Autoxidation of MPDP + into MPP + was monitored photospectrometrically. (E, F) MPDP + (100 μM) was kept under atmospheric (E) or oxygen-free conditions (F) in potassium phosphate buffer of different pH at 37°C for the indicated incubation intervals. (G, H) To identify the influence of metabolites of the dopamine pathway and physiologically relevant iron-containing molecules, MPDP + (100 μM) was treated with the indicated compounds (100 μM each) under aerobic conditions with a pH of 7.4. All data are mean ± SD ( n = 8) * p
    Dmem, supplied by AddexBio, used in various techniques. Bioz Stars score: 97/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Avantor dmem
    Modulation of the nonenzymatic conversion of MPDP + into MPP + . (A) MPDP + (100 μM) was kept under atmospheric conditions in <t>DMEM</t> medium at <t>37°C</t> or under oxygen-free conditions (B) for the time intervals indicated. MPP + , MPDP + , and MPTP were detected by HPLC. (C, D) MPDP + (100 μM) was maintained in potassium phosphate buffer with varying pH, as indicated in each time interval. Autoxidation of MPDP + into MPP + was monitored photospectrometrically. (E, F) MPDP + (100 μM) was kept under atmospheric (E) or oxygen-free conditions (F) in potassium phosphate buffer of different pH at 37°C for the indicated incubation intervals. (G, H) To identify the influence of metabolites of the dopamine pathway and physiologically relevant iron-containing molecules, MPDP + (100 μM) was treated with the indicated compounds (100 μM each) under aerobic conditions with a pH of 7.4. All data are mean ± SD ( n = 8) * p
    Dmem, supplied by Avantor, used in various techniques. Bioz Stars score: 97/100, based on 292 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Becton Dickinson dmem
    Modulation of the nonenzymatic conversion of MPDP + into MPP + . (A) MPDP + (100 μM) was kept under atmospheric conditions in <t>DMEM</t> medium at <t>37°C</t> or under oxygen-free conditions (B) for the time intervals indicated. MPP + , MPDP + , and MPTP were detected by HPLC. (C, D) MPDP + (100 μM) was maintained in potassium phosphate buffer with varying pH, as indicated in each time interval. Autoxidation of MPDP + into MPP + was monitored photospectrometrically. (E, F) MPDP + (100 μM) was kept under atmospheric (E) or oxygen-free conditions (F) in potassium phosphate buffer of different pH at 37°C for the indicated incubation intervals. (G, H) To identify the influence of metabolites of the dopamine pathway and physiologically relevant iron-containing molecules, MPDP + (100 μM) was treated with the indicated compounds (100 μM each) under aerobic conditions with a pH of 7.4. All data are mean ± SD ( n = 8) * p
    Dmem, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 97/100, based on 4854 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Biochrom dmem
    <t>RPE</t> cell viability was unaffected by galectin-1. Human RPE cells were seeded in 96-well plates at an initial density of 1×10 4 cells/well in <t>DMEM</t> supplemented with 10% FCS. Cells were cultured overnight before the indicated concentrations of galectin-1 were added to the medium in DMEM containing 2% FCS, and cell survival was assessed by MTT assay after 24 h ( A ) and 48 h ( B ) of culture. RPE cells cultured with incubation medium alone served as controls. The values were normalized to untreated controls (100%) and represent the mean±SD of six experiments performed in duplicate wells.
    Dmem, supplied by Biochrom, used in various techniques. Bioz Stars score: 97/100, based on 1490 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Biological Industries Inc dmem
    Effect of OA-GL12 on the repair rate of HaCaT cells scratches HaCaT cells (2.5 × 10 5 /well) were cultured in 24-well plates for 12–24 h with serum starvation and then made to the mechanical scratch wound by a sterile pipette tip. After washed, cells were cultured for next periods (from 0 to 24 h) in a serum-free basal medium with the continued presence of OA-GL12. The same volume of <t>DMEM</t> (serum free) was added as vehicle and OM-LV20 (50 nM) as positive control. ( A ). OA-GL12 (10 pM) showed prominent wound-healing capacity on HaCaT cells. ( B ). OA-GL12 illustrated time- and concentration- dependent wound-healing capacity on HaCaT cells. Contain cells were added into 96-well plates with basic DMEM (90 μl) without 10% <t>FBS</t> and incubated for 2-4 h. Afterward, OA-GL12 (10 μl) was added to each well and incubated for 24 h at various final concentrations then tested using a CellTiter 96® AQueous One Solution Assay. ( C ). OM-LV20 (50 nM) showed proliferative effect, but OA-GL12 did not show on HaCaT cells from 2 to 10 pM. Data were presented as mean ± S.D., n =9. * P
    Dmem, supplied by Biological Industries Inc, used in various techniques. Bioz Stars score: 97/100, based on 1043 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Biowest SAS dmem
    <t>EA.hy926</t> cells secretome induce LOX-1 shedding. (A) Western blot analysis of conditioned medium derived from cross-cells experiments. LOX-1-V5 COS transfected cells were incubated for 10 min with <t>DMEM</t> (lane 1), medium derived from untreated EA cells (lane 2) and medium derived from EA cells conditioned in the presence of ox-LDL (20μg/ml) (lane 3). The histogram on the right shows the densitometric analysis of sLOX-1 band intensity. (B) Effect of different classes of protease inhibitors on LOX-1 shedding. Inhibitors were added during the 10 min final incubation time. GM6001 was used at 2.6 and 26μM (lanes 3 and 4), Pepstatin A at 10μM (lane 5) and PMSF at 2mM (lane 6).
    Dmem, supplied by Biowest SAS, used in various techniques. Bioz Stars score: 97/100, based on 515 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Caisson Labs dmem
    <t>EA.hy926</t> cells secretome induce LOX-1 shedding. (A) Western blot analysis of conditioned medium derived from cross-cells experiments. LOX-1-V5 COS transfected cells were incubated for 10 min with <t>DMEM</t> (lane 1), medium derived from untreated EA cells (lane 2) and medium derived from EA cells conditioned in the presence of ox-LDL (20μg/ml) (lane 3). The histogram on the right shows the densitometric analysis of sLOX-1 band intensity. (B) Effect of different classes of protease inhibitors on LOX-1 shedding. Inhibitors were added during the 10 min final incubation time. GM6001 was used at 2.6 and 26μM (lanes 3 and 4), Pepstatin A at 10μM (lane 5) and PMSF at 2mM (lane 6).
    Dmem, supplied by Caisson Labs, used in various techniques. Bioz Stars score: 97/100, based on 70 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Cambrex dmem
    <t>EA.hy926</t> cells secretome induce LOX-1 shedding. (A) Western blot analysis of conditioned medium derived from cross-cells experiments. LOX-1-V5 COS transfected cells were incubated for 10 min with <t>DMEM</t> (lane 1), medium derived from untreated EA cells (lane 2) and medium derived from EA cells conditioned in the presence of ox-LDL (20μg/ml) (lane 3). The histogram on the right shows the densitometric analysis of sLOX-1 band intensity. (B) Effect of different classes of protease inhibitors on LOX-1 shedding. Inhibitors were added during the 10 min final incubation time. GM6001 was used at 2.6 and 26μM (lanes 3 and 4), Pepstatin A at 10μM (lane 5) and PMSF at 2mM (lane 6).
    Dmem, supplied by Cambrex, used in various techniques. Bioz Stars score: 96/100, based on 629 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Cell Signaling Technology Inc dmem
    <t>EA.hy926</t> cells secretome induce LOX-1 shedding. (A) Western blot analysis of conditioned medium derived from cross-cells experiments. LOX-1-V5 COS transfected cells were incubated for 10 min with <t>DMEM</t> (lane 1), medium derived from untreated EA cells (lane 2) and medium derived from EA cells conditioned in the presence of ox-LDL (20μg/ml) (lane 3). The histogram on the right shows the densitometric analysis of sLOX-1 band intensity. (B) Effect of different classes of protease inhibitors on LOX-1 shedding. Inhibitors were added during the 10 min final incubation time. GM6001 was used at 2.6 and 26μM (lanes 3 and 4), Pepstatin A at 10μM (lane 5) and PMSF at 2mM (lane 6).
    Dmem, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 333 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Cellgro dmem
    Relationship between the incidence of PC and rate of proliferation in 184A1 and <t>MDA-MB-231.</t> ( A,B ) 184A1 was cultured in three different media: <t>DMEM</t> without FBS (D S−), DMEM with 10% FBS (D S+), and optimal basal medium with supplements and growth factors (MEM). PC were labeled by anti-acetylated α-tubulin (red) and proliferating cells by anti-Ki-67 (green). ( A ) PC were located only in non-proliferating Ki-67-negative cells. ( B ) The incidence of PC (red bars) in 184A1 cells was higher in serum-free conditions (D S−) and with longer duration of culture. Data are the mean and standard error of two independent experiments performed in triplicate. ( C ) MDA-MB-231 were grown in DMEM with (D S+) and without (D S−) serum. Proliferation (Ki-67 labeling, green bars) was lower in serum-free conditions; however, there was no corresponding statistically significant increase in PC (red bars). Data are the mean of two independent experiments performed in triplicate. Bar: A = 10 μm.
    Dmem, supplied by Cellgro, used in various techniques. Bioz Stars score: 97/100, based on 4107 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Corning Life Sciences dmem
    Effects of amino acids on ERK phosphorylation Panels A and B: Western blot demonstrating the time course of ERK phosphorylation elicited by meat extract a mixture of amino acids. <t>BMSCs</t> from C57BL/6 mice were grown in 60 mm 2 dishes in <t>DMEM</t> + 10% fetal calf serum to ~ 80% confluency, then switched to KRB buffer +0.2% BSA. After an overnight incubation, cells were stimulated with a 0.3% meat extract for the indicated times. Cellular protein was extracted and 50 μg/lane was separated by electrophoresis on SDS-PAGE gels. For Western blotting with ECL detection, antibodies directed against both total ERK and phospho-ERK were utilized. Primary (1:750, 4°C, overnight) and HRP-coupled secondary (1:2000, room temperature, 1 hour) antibodies were obtained from Cell Signaling Technology (Danvers, MA). Phorbol ester (TPA, 1 μM, 10 min) was used as a positive control. (A) Representative blot. (B) Quantitation by densitometric analysis of six different experiments from three different cell preparations. Values are means±SEMs. *P
    Dmem, supplied by Corning Life Sciences, used in various techniques. Bioz Stars score: 97/100, based on 4969 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    EuroClone dmem
    SCD1 is needed for <t>MCF-7</t> and MDA-MB-231 cell migration. ( A ) Pharmacological inactivation of SCD1 with the small-molecule inhibitor A939572 decreased the migratory ability of both MCF-7 and MDA-MB-231 cells in an in vitro wound-healing assay. Cells were treated with 1 μ M A939572 or with vehicle (control, ctrl) in serum-free <t>DMEM</t> for 24–48 h prior to assay. ( B ) siRNA-mediated knockdown of SCD1 reduced MCF-7 and MDA-MB-231 migration in an in vitro wound-healing assay. Cell were transiently transfected for 72 h with 60 pmol of either siRNA ctrl and siRNA SCD1 prior to assay. ( A , B ) Cell proliferation was prevented by a 2 h pretreatment with mitomycin C (5 μ g ml −1 ). A vertical scratch was made with a pipette tip in the confluent cell monolayers and cell motility assayed at the indicated times ( x -axis). The gaps were photographed at 0, 24 and 48 h after wounding (original magnification × 100) and the distance migrated was measured in micrometres ( y -axis) relative to zero time using ImageJ software. All experiments were run in triplicate and repeated three times. The data shown are the mean±s.e. * P
    Dmem, supplied by EuroClone, used in various techniques. Bioz Stars score: 97/100, based on 874 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Fisher Scientific dmem
    SCD1 is needed for <t>MCF-7</t> and MDA-MB-231 cell migration. ( A ) Pharmacological inactivation of SCD1 with the small-molecule inhibitor A939572 decreased the migratory ability of both MCF-7 and MDA-MB-231 cells in an in vitro wound-healing assay. Cells were treated with 1 μ M A939572 or with vehicle (control, ctrl) in serum-free <t>DMEM</t> for 24–48 h prior to assay. ( B ) siRNA-mediated knockdown of SCD1 reduced MCF-7 and MDA-MB-231 migration in an in vitro wound-healing assay. Cell were transiently transfected for 72 h with 60 pmol of either siRNA ctrl and siRNA SCD1 prior to assay. ( A , B ) Cell proliferation was prevented by a 2 h pretreatment with mitomycin C (5 μ g ml −1 ). A vertical scratch was made with a pipette tip in the confluent cell monolayers and cell motility assayed at the indicated times ( x -axis). The gaps were photographed at 0, 24 and 48 h after wounding (original magnification × 100) and the distance migrated was measured in micrometres ( y -axis) relative to zero time using ImageJ software. All experiments were run in triplicate and repeated three times. The data shown are the mean±s.e. * P
    Dmem, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 97/100, based on 765 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    FUJIFILM dmem
    The LRS of SQSTM1 and TBC1D25 is essential for phagophore localization, but not sufficient for their degradation. (A) Schematic representation of the LRS-swapping mutants of TBC1D25 and SQSTM1, named TBC1D25-(SQT-LRS) and SQSTM1-(TBC-LRS), respectively. The asterisks indicate the residues that are highly conserved in many LRSs. The WA mutant contains a Trp-to-Ala (WA) mutation (shaded background), which disrupts binding activity to LC3 homologs. (B) Differing LC3 homolog binding activity of TBC1D25, TBC1D25-W136A (designated as WA), and TBC1D25-(SQT-LRS) (designated as SQT) as revealed by GST affinity isolation assays. COS-7 cell lysates expressing EGFP-TBC1D25, EGFP-TBC1D25-WA, or EGFP-TBC1D25-(SQT-LRS) were incubated with GST-LC3, GST-GABARAP, or GST-GABARAPL2 that had been coupled with glutathione-Sepharose beads. Proteins bound to the beads were analyzed by immunoblotting with HRP-conjugated anti-GFP antibody (top panel) and HRP-conjugated anti-GST antibody (bottom panel). The asterisks indicate the nonspecific bands of the anti-GFP antibody. (C) sqstm1 -KO <t>MEF</t> cells stably expressing FLAG-SQSTM1, FLAG-SQSTM1-WA, FLAG-SQSTM1-(TBC-LRS) (FLAG-SQSTM1; left panels), FLAG-TBC1D25, FLAG-TBC1D25-WA, or FLAG-TBC1D25-(SQT-LRS) (FLAG-TBC1D25; right panels) were cultured for 2 h in <t>DMEM</t> (nutrient-rich; - starvation) or HBSS (starvation) with or without 20 μM BafA1. Cell lysates were analyzed by immunoblotting with anti-FLAG tag antibody (top panels) and anti-ACTB antibody (bottom panels). The positions of the molecular mass markers (in kilodaltons) are shown on the left.
    Dmem, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 97/100, based on 1473 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare dmem
    Metabolic inhibition of sulfation of HS chains leads to a loss of sulfated groups of HS, Hsp90α, and Hsp90β from the cell surface ( a ) and to an inhibition of the basal and Hsp90-stimulated cell migration and invasion ( b – d ). a Cells were treated with sodium chlorate, after which cells were grown at 37 °C in <t>DMEM-FBS</t> without chlorate. At indicated times, cell surface HS and proteins were stained with specific antibodies, analyzed by flow cytometry, and quantified. The data are presented as the MFI specific for HS, Hsp90α, Hsp90β, and LRP1, expressed in percent. The specific MFI of control untreated cells was taken as 100%. b The migration of chlorate-treated cells in the wound-healing assay in the presence and absence of Hsp90. c The analysis of the basal migration/invasion of chlorate-treated cells. The basal migration/invasion of cells expressed in percent is presented. The basal migration/invasion of untreated control cells was taken as 100%. d ” section and expressed in percent. The Hsp90-stimulated migration/invasion of control untreated cells was taken as 100%. a , c , d Each bar represents the mean ± SD ( n = 3–4). * p
    Dmem, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 97/100, based on 16922 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Gemini Bio dmem
    Metabolic inhibition of sulfation of HS chains leads to a loss of sulfated groups of HS, Hsp90α, and Hsp90β from the cell surface ( a ) and to an inhibition of the basal and Hsp90-stimulated cell migration and invasion ( b – d ). a Cells were treated with sodium chlorate, after which cells were grown at 37 °C in <t>DMEM-FBS</t> without chlorate. At indicated times, cell surface HS and proteins were stained with specific antibodies, analyzed by flow cytometry, and quantified. The data are presented as the MFI specific for HS, Hsp90α, Hsp90β, and LRP1, expressed in percent. The specific MFI of control untreated cells was taken as 100%. b The migration of chlorate-treated cells in the wound-healing assay in the presence and absence of Hsp90. c The analysis of the basal migration/invasion of chlorate-treated cells. The basal migration/invasion of cells expressed in percent is presented. The basal migration/invasion of untreated control cells was taken as 100%. d ” section and expressed in percent. The Hsp90-stimulated migration/invasion of control untreated cells was taken as 100%. a , c , d Each bar represents the mean ± SD ( n = 3–4). * p
    Dmem, supplied by Gemini Bio, used in various techniques. Bioz Stars score: 97/100, based on 748 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    HiMedia Laboratories dmem
    Haemocytometer and Microscopy based <t>HeLa</t> Cell Proliferation Data is Presented. ( Figure 2A ) represents the photomicrograph taken at 10X of HeLa growing in six well plate with different treatment condition whereas <t>DMEM</t> Control, EFM: DMEM and BCEF: DMEM. The Microscopy photographs were taken at the end of treatment at 48 h. The Microscopy photographs were captured using light inverted microscope. ( Figure 2B ) is a graphical representative oftotal cells after treatment condition (DMEM Control DMEM Control, EFM: DMEM and BCEF: DMEM). The Total cells were recorded using Haemocytometer and data is presented as Percentage of Total Cell Count X 10 4 in each treatment conditions. The Data are represented as Mean ± SD. Each experiment was conducted independently four times. ( Figure 2C ) shows us the percent viability of cells after the respective treatment conditions. ( Figure 2D ) The photograph depicts the photomicrograph taken at 10X for HeLa cell growing in 96 Well Plate with different treatment condition (DMEM Control, EFM: DMEM and BCEF: DMEM). The microscopy photographs were taken at the end of MTT based cytotoxicity assay showing Formazan crystals formed, using 10 X Objective. ( Figure 2E ) Graphical representation of absorbance captured by ELISA plate reader with cells treated with DMEM Control DMEM Control, EFM: DMEM and BCEF: DMEM. Total cells were recorded by taking absorbance with ELISA reader. The Data are represented as Mean ± SD. Each experiment was conducted independently three times.
    Dmem, supplied by HiMedia Laboratories, used in various techniques. Bioz Stars score: 97/100, based on 335 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Irvine Scientific dmem
    Haemocytometer and Microscopy based <t>HeLa</t> Cell Proliferation Data is Presented. ( Figure 2A ) represents the photomicrograph taken at 10X of HeLa growing in six well plate with different treatment condition whereas <t>DMEM</t> Control, EFM: DMEM and BCEF: DMEM. The Microscopy photographs were taken at the end of treatment at 48 h. The Microscopy photographs were captured using light inverted microscope. ( Figure 2B ) is a graphical representative oftotal cells after treatment condition (DMEM Control DMEM Control, EFM: DMEM and BCEF: DMEM). The Total cells were recorded using Haemocytometer and data is presented as Percentage of Total Cell Count X 10 4 in each treatment conditions. The Data are represented as Mean ± SD. Each experiment was conducted independently four times. ( Figure 2C ) shows us the percent viability of cells after the respective treatment conditions. ( Figure 2D ) The photograph depicts the photomicrograph taken at 10X for HeLa cell growing in 96 Well Plate with different treatment condition (DMEM Control, EFM: DMEM and BCEF: DMEM). The microscopy photographs were taken at the end of MTT based cytotoxicity assay showing Formazan crystals formed, using 10 X Objective. ( Figure 2E ) Graphical representation of absorbance captured by ELISA plate reader with cells treated with DMEM Control DMEM Control, EFM: DMEM and BCEF: DMEM. Total cells were recorded by taking absorbance with ELISA reader. The Data are represented as Mean ± SD. Each experiment was conducted independently three times.
    Dmem, supplied by Irvine Scientific, used in various techniques. Bioz Stars score: 96/100, based on 269 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    dmem  (Lonza)
    97
    Lonza dmem
    THS extracts made in basal <t>DMEM</t> without serum proteins showed little cytotoxicity to <t>mNSC.</t> (A–D) Extracts were prepared from terry cloth exposed to cigarette smoke for different times and tested with mNSC in the MTT assay. (E) Dose response curves showing the cytotoxicity of THS extracts prepared from terry cloth exposed to 54 cigarettes over 8 months with and without serum proteins. Each curve represents mean ± SEM of three experiments. * = p
    Dmem, supplied by Lonza, used in various techniques. Bioz Stars score: 97/100, based on 5648 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Mediatech dmem
    Effect of IBTP treatment on mitochondrial respiration of <t>MB231</t> cells. Panel A: Cells plated on XF24 plates were treated with the indicated concentrations of IBTP or BTPP for 4h in 0.5% FBS-containing medium. After treatment, the medium was removed and replaced with XF assay medium <t>(DMEM,</t> containing 5mM glucose, 0.5% FBS, 5mM HEPES without bicarbonate) and equilibrated 1h before OCR measurement. Panel B: Cells plated on 6-well plates were treated with the indicated concentrations of IBTP or BTPP for 4h. After the incubation, the cells were harvested immediately by trypsinization. The harvested cells were replated in XF24 plates and allowed adhere for an additional 20h in complete medium containing 10% FBS (total 24h). The medium was removed and replaced with assay medium and equilibrated 1h before OCR measurement. Panel C : After 4h of IBTP or BTPP treatment, the medium was replaced with complete medium containing 10% FBS, and incubated for 48h. The cells were harvested after 48h, replated in XF24 plates and allowed adhere for an additional 20h in complete medium. The medium was replaced with assay media and incubated 1h before measurement of OCR (total duration 72h).
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    Merck & Co dmem
    Effect of IBTP treatment on mitochondrial respiration of <t>MB231</t> cells. Panel A: Cells plated on XF24 plates were treated with the indicated concentrations of IBTP or BTPP for 4h in 0.5% FBS-containing medium. After treatment, the medium was removed and replaced with XF assay medium <t>(DMEM,</t> containing 5mM glucose, 0.5% FBS, 5mM HEPES without bicarbonate) and equilibrated 1h before OCR measurement. Panel B: Cells plated on 6-well plates were treated with the indicated concentrations of IBTP or BTPP for 4h. After the incubation, the cells were harvested immediately by trypsinization. The harvested cells were replated in XF24 plates and allowed adhere for an additional 20h in complete medium containing 10% FBS (total 24h). The medium was removed and replaced with assay medium and equilibrated 1h before OCR measurement. Panel C : After 4h of IBTP or BTPP treatment, the medium was replaced with complete medium containing 10% FBS, and incubated for 48h. The cells were harvested after 48h, replated in XF24 plates and allowed adhere for an additional 20h in complete medium. The medium was replaced with assay media and incubated 1h before measurement of OCR (total duration 72h).
    Dmem, supplied by Merck & Co, used in various techniques. Bioz Stars score: 97/100, based on 167 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Merck KGaA dmem
    Effect of IBTP treatment on mitochondrial respiration of <t>MB231</t> cells. Panel A: Cells plated on XF24 plates were treated with the indicated concentrations of IBTP or BTPP for 4h in 0.5% FBS-containing medium. After treatment, the medium was removed and replaced with XF assay medium <t>(DMEM,</t> containing 5mM glucose, 0.5% FBS, 5mM HEPES without bicarbonate) and equilibrated 1h before OCR measurement. Panel B: Cells plated on 6-well plates were treated with the indicated concentrations of IBTP or BTPP for 4h. After the incubation, the cells were harvested immediately by trypsinization. The harvested cells were replated in XF24 plates and allowed adhere for an additional 20h in complete medium containing 10% FBS (total 24h). The medium was removed and replaced with assay medium and equilibrated 1h before OCR measurement. Panel C : After 4h of IBTP or BTPP treatment, the medium was replaced with complete medium containing 10% FBS, and incubated for 48h. The cells were harvested after 48h, replated in XF24 plates and allowed adhere for an additional 20h in complete medium. The medium was replaced with assay media and incubated 1h before measurement of OCR (total duration 72h).
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    Millipore dmem
    Sequence comparison of ECs1581 variants in E. coli . A. Multiple alignment of the predicted amino acid sequences of ECs1581 in different lineages of O157:H7. Residue divergence specific to lineage I EHEC O157:H7 isolates is marked by a pink shaded box. B. Multiple alignment of EHEC variant ECs1581 (EDL933/Sakai) with commensal variant ECED1_1787 (ED1a) and UPEC variant C1493 (CFT073). The RGD motif is at amino acid position 20–22. Residue divergence between the strains is indicated. C. SDS-PAGE gel showing stimulation of <t>T3S</t> in an EHEC Δ ecs1581 mutant expressing EHEC ECs1581 (pECs1581) or UPEC C1493 (pC1493) from a low copy number plasmid. pWControl alone in the ecs1581 mutant was used as a negative control strain. TCA-precipitated culture supernatants were analysed as described previously in Experimental procedures . D. Analysis of motility repression in an EHEC Δ ecs1581 and UPEC Δ c1493 deletion background expressing pECs1581, pC1493 or pWControl. Motility was assessed as described in Experimental procedures . E. SDS-PAGE gel showing T3S profile for EHEC strain ZAP193 and isogenic Δ grlA ::Tn mutant expressing pECs1581 or pWControl. Strains were cultured in <t>DMEM</t> to an OD 600 of 1.0 and culture supernatant proteins were analysed exactly as described for strains grown in MEM-HEPES.
    Dmem, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 49883 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Nacalai dmem
    Tim4-affinity purification of sEVs. ( a ) Thioglycollate-elicited mouse peritoneal macrophages <t>(pMac)</t> (3 × 10 5 cells/ml) were cultured in 10 ml of <t>DMEM</t> containing 5% EV-depleted FBS for 48 h. 10K sup of pMac (4 ml each) were incubated with Dynabeads or mouse Tim4-Fc-conjugated Dynabeads overnight. Bound sEVs were eluted, subjected to SDS-PAGE and analyzed by western blot (right panel) with anti-CD63, anti-CD9, or anti-CD81 antibody. Total proteins in the sEV fractions were visualized by Oriole fluorescent gel stain (left panel). The amount loaded in the input lane was equivalent to 1/1000 volume of the total supernatant. ( b , c ) sEVs were purified from 10K sup (4 ml of medium containing 5% EV-depleted FBS) of K562 cells (1 × 10 6 cells/ml) ( b ) or pMac (4 × 10 5 cells/ml) ( c ) by each method. The purified sEV fractions were adjusted to the same volume (60 μl), subjected to SDS-PAGE, and analyzed by Oriole stain (left panels) or western blot (right panels). TEI: Total Exosome Isolation. The number of sEV particles in the purified fractions were also analyzed by NTA assay using NanoSight LM10 (Malvern), and the calculated purification recovery yield relative to total sEV particles was plotted in the lower graphs. ( d , e ) Protein concentration of purified sEV fractions from K562 cells ( d ) or pMac ( e ) was determined by BCA protein assay, and adjusted to the same in each method. Equal amount of proteins (100 ng) were subjected to SDS-PAGE, and analyzed by Oriole stain (left panels) or western blot (right panels). ( f ) The flow through of 10K sup of K562 cells after Tim4-affinity purification was further ultracentrifuged, and the same proportion of the protein amount to the total purified protein amount as Tim4-affinity purification was loaded to detect residual exosomes by western blot with anti-CD63 antibody (Tim4 FT → UC). ( g , h ) sEVs were isolated from 50 μl of mouse serum ( g ) or 500 μl of human urine ( h ) by each method, adjusted to the same volume and analyzed by western blot.
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    Image Search Results


    CEP350 is a melanoma tumor suppressor. ( a ) CEP350 alterations encoded in human melanoma genomes. Amino acid positions in gray, black and red text denote synonymous, nonsynonymous and non-damaging, and nonsynonymous and damaging alterations, respectively. Single and double asterisks denote 2 genomes with ≥2 mutations. ( b–e ) CEP350 depletion accelerates xenograft progression in vivo . Primary shRNA experiment consisting of 1 million A375 cells stably expressing control shNTC construct (MOI of 6; n = 10) or 3 non-overlapping, pooled shRNA constructs for CEP350 (clones 689, 691 and 694, each at MOI of 2; n = 10) injected subcutaneously into immunodeficient NSG mice. ( b ) Xenograft volumes calculated over a 1-month period. ( c ) The cohorts receiving the CEP350 shRNA pool grew significantly faster than matched cohorts receiving control shNTC. The growth curves differed significantly ( P

    Journal: Nature genetics

    Article Title: Transposon mutagenesis identifies genetic drivers of BrafV600E melanoma

    doi: 10.1038/ng.3275

    Figure Lengend Snippet: CEP350 is a melanoma tumor suppressor. ( a ) CEP350 alterations encoded in human melanoma genomes. Amino acid positions in gray, black and red text denote synonymous, nonsynonymous and non-damaging, and nonsynonymous and damaging alterations, respectively. Single and double asterisks denote 2 genomes with ≥2 mutations. ( b–e ) CEP350 depletion accelerates xenograft progression in vivo . Primary shRNA experiment consisting of 1 million A375 cells stably expressing control shNTC construct (MOI of 6; n = 10) or 3 non-overlapping, pooled shRNA constructs for CEP350 (clones 689, 691 and 694, each at MOI of 2; n = 10) injected subcutaneously into immunodeficient NSG mice. ( b ) Xenograft volumes calculated over a 1-month period. ( c ) The cohorts receiving the CEP350 shRNA pool grew significantly faster than matched cohorts receiving control shNTC. The growth curves differed significantly ( P

    Article Snippet: The human melanoma cell lines A375 (CRL-1619) and COLO-829 (CRL-1974) were purchased from the American Type Culture Collection (ATCC) and grown according to the manufacturer’s specifications in complete medium (1× DMEM (ATCC, 30-2002) for A375 cells or 1× RPMI-1640 medium (ATCC, 30-2001) for COLO829 cells) supplemented with 10% FBS and 1× penicillin-streptomycin grown at 37 °C in 5% CO2 .

    Techniques: In Vivo, shRNA, Stable Transfection, Expressing, Construct, Injection, Mouse Assay

    Morphological characteristics and functional analysis of cell line–derived ESCC 3D organoids. ESCC 3D organoids were grown with TE11 cells in indicated cell culture media to acquire ( A ) phase contrast images and evaluate ( B ) organoid growth (size) at indicated time points. ( C ) Organoid formation rate was determined at day 11. RPMI1640 and DMEM are supplemented with 10% FBS. aDMEM/F12 + is fully supplemented as used to grow patient-derived 3D organoids. Scale bar =100 μm. ( B ) Data are presented as mean ± SD of at least 7 organoids. Data represent 3 independent experiments with similar results and 1-way analysis of variance with multiple comparisons (Tukey) was performed for each ( B ) time point in and ( C ) condition. * P

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: Three-Dimensional Organoids Reveal Therapy Resistance of Esophageal and Oropharyngeal Squamous Cell Carcinoma Cells

    doi: 10.1016/j.jcmgh.2018.09.003

    Figure Lengend Snippet: Morphological characteristics and functional analysis of cell line–derived ESCC 3D organoids. ESCC 3D organoids were grown with TE11 cells in indicated cell culture media to acquire ( A ) phase contrast images and evaluate ( B ) organoid growth (size) at indicated time points. ( C ) Organoid formation rate was determined at day 11. RPMI1640 and DMEM are supplemented with 10% FBS. aDMEM/F12 + is fully supplemented as used to grow patient-derived 3D organoids. Scale bar =100 μm. ( B ) Data are presented as mean ± SD of at least 7 organoids. Data represent 3 independent experiments with similar results and 1-way analysis of variance with multiple comparisons (Tukey) was performed for each ( B ) time point in and ( C ) condition. * P

    Article Snippet: Cells were then resuspended in 1-mL advanced Dulbecco’s modified Eagle medium (DMEM)/F12 (12491015, Thermo Fisher Scientific) containing 100-U/mL penicillin and 100-μg/mL streptomycin (15140122, Thermo Fisher Scientific), 1×GlutaMAX (35050061, Thermo Fisher Scientific), and 1×HEPES (15630080, Thermo Fisher Scientific) (aDMEM/F12+ ).

    Techniques: Functional Assay, Derivative Assay, Cell Culture

    Modulation of the nonenzymatic conversion of MPDP + into MPP + . (A) MPDP + (100 μM) was kept under atmospheric conditions in DMEM medium at 37°C or under oxygen-free conditions (B) for the time intervals indicated. MPP + , MPDP + , and MPTP were detected by HPLC. (C, D) MPDP + (100 μM) was maintained in potassium phosphate buffer with varying pH, as indicated in each time interval. Autoxidation of MPDP + into MPP + was monitored photospectrometrically. (E, F) MPDP + (100 μM) was kept under atmospheric (E) or oxygen-free conditions (F) in potassium phosphate buffer of different pH at 37°C for the indicated incubation intervals. (G, H) To identify the influence of metabolites of the dopamine pathway and physiologically relevant iron-containing molecules, MPDP + (100 μM) was treated with the indicated compounds (100 μM each) under aerobic conditions with a pH of 7.4. All data are mean ± SD ( n = 8) * p

    Journal: Antioxidants & Redox Signaling

    Article Title: Preferential Extracellular Generation of the Active Parkinsonian Toxin MPP+ by Transporter-Independent Export of the Intermediate MPDP+

    doi: 10.1089/ars.2015.6297

    Figure Lengend Snippet: Modulation of the nonenzymatic conversion of MPDP + into MPP + . (A) MPDP + (100 μM) was kept under atmospheric conditions in DMEM medium at 37°C or under oxygen-free conditions (B) for the time intervals indicated. MPP + , MPDP + , and MPTP were detected by HPLC. (C, D) MPDP + (100 μM) was maintained in potassium phosphate buffer with varying pH, as indicated in each time interval. Autoxidation of MPDP + into MPP + was monitored photospectrometrically. (E, F) MPDP + (100 μM) was kept under atmospheric (E) or oxygen-free conditions (F) in potassium phosphate buffer of different pH at 37°C for the indicated incubation intervals. (G, H) To identify the influence of metabolites of the dopamine pathway and physiologically relevant iron-containing molecules, MPDP + (100 μM) was treated with the indicated compounds (100 μM each) under aerobic conditions with a pH of 7.4. All data are mean ± SD ( n = 8) * p

    Article Snippet: These IMAs were cultured at 37°C (5% CO2 ) in DMEM (normal glucose) (GIBCO) with 10% FCS (fetal calf serum from PAA) and 1% penicillin/streptomycin (10,000 U/ml stock).

    Techniques: High Performance Liquid Chromatography, Incubation

    Populations of PDPN+ cells among CAFs affected by environmental conditions. (A) The percentage of PDPN+ cells among CAFs changed in a time-dependent manner by culturing with DMEM-containing growth factors. (B) Populations of PDPN+ cells among CAFs changed by the addition of FBS in concentration- and time-dependent manners. The control (CTRL) condition consisted of DMEM containing 10% FBS and medium changes every day. (C) Populations of PDPN+ cells in cultures of serum-free DMEM increased more rapidly than those in cultures of glucose- and serum-free DMEM.

    Journal: Molecular Cancer

    Article Title: Podoplanin expression in cancer-associated fibroblasts enhances tumor progression of invasive ductal carcinoma of the pancreas

    doi: 10.1186/1476-4598-12-168

    Figure Lengend Snippet: Populations of PDPN+ cells among CAFs affected by environmental conditions. (A) The percentage of PDPN+ cells among CAFs changed in a time-dependent manner by culturing with DMEM-containing growth factors. (B) Populations of PDPN+ cells among CAFs changed by the addition of FBS in concentration- and time-dependent manners. The control (CTRL) condition consisted of DMEM containing 10% FBS and medium changes every day. (C) Populations of PDPN+ cells in cultures of serum-free DMEM increased more rapidly than those in cultures of glucose- and serum-free DMEM.

    Article Snippet: In the PDPN induction assay, DMEM (Sigma Chemical Co., St. Louis, MO) or DMEM containing no glucose (Invitrogen, Carlsbad, CA) was used for cell culture.

    Techniques: Concentration Assay

    RPE cell viability was unaffected by galectin-1. Human RPE cells were seeded in 96-well plates at an initial density of 1×10 4 cells/well in DMEM supplemented with 10% FCS. Cells were cultured overnight before the indicated concentrations of galectin-1 were added to the medium in DMEM containing 2% FCS, and cell survival was assessed by MTT assay after 24 h ( A ) and 48 h ( B ) of culture. RPE cells cultured with incubation medium alone served as controls. The values were normalized to untreated controls (100%) and represent the mean±SD of six experiments performed in duplicate wells.

    Journal: Molecular Vision

    Article Title: Inhibition of human retinal pigment epithelial cell attachment, spreading, and migration by the human lectin galectin-1

    doi:

    Figure Lengend Snippet: RPE cell viability was unaffected by galectin-1. Human RPE cells were seeded in 96-well plates at an initial density of 1×10 4 cells/well in DMEM supplemented with 10% FCS. Cells were cultured overnight before the indicated concentrations of galectin-1 were added to the medium in DMEM containing 2% FCS, and cell survival was assessed by MTT assay after 24 h ( A ) and 48 h ( B ) of culture. RPE cells cultured with incubation medium alone served as controls. The values were normalized to untreated controls (100%) and represent the mean±SD of six experiments performed in duplicate wells.

    Article Snippet: After reaching confluency, primary RPE cells were subcultured and maintained in DMEM (Biochrom) supplemented with 10% FCS (Biochrom) at 37 °C and 5% CO2 .

    Techniques: Cell Culture, MTT Assay, Incubation

    Galectin-1 inhibits spreading of RPE cells on fibronectin and laminin. A, B : RPE suspensions were incubated for 35 min with the indicated concentrations of galectin-1 and then plated on four chamber slides coated with either Fn ( A ) or Lam ( B ) at a density of 5×10 4 cells per chamber. Cells were allowed to spread in DMEM for 3.5 h at 37 °C before fixation and Giemsa staining. Values indicate means±SD of four experiments performed in duplicate and are expressed as percentage of controls without galectin-1 in the medium. ( C ) RPE cell spreading on galectin-1 as a substrate. Four chamber slides were coated with either Fn alone, Fn+galectin-1 (Gal-1), Lam alone, Lam+Gal-1, or Gal-1 alone. RPE cells were then plated at a density of 5×10 4 cells per chamber and allowed to spread. Results were obtained from evaluation of four separate fields by examining at least 100 cells per field. Values indicate means±SD of three experiments performed in duplicate and are expressed as percentage of cells spreading on Fn or Lam as a substrate. Statistical analysis was performed using Students t -test and a p value

    Journal: Molecular Vision

    Article Title: Inhibition of human retinal pigment epithelial cell attachment, spreading, and migration by the human lectin galectin-1

    doi:

    Figure Lengend Snippet: Galectin-1 inhibits spreading of RPE cells on fibronectin and laminin. A, B : RPE suspensions were incubated for 35 min with the indicated concentrations of galectin-1 and then plated on four chamber slides coated with either Fn ( A ) or Lam ( B ) at a density of 5×10 4 cells per chamber. Cells were allowed to spread in DMEM for 3.5 h at 37 °C before fixation and Giemsa staining. Values indicate means±SD of four experiments performed in duplicate and are expressed as percentage of controls without galectin-1 in the medium. ( C ) RPE cell spreading on galectin-1 as a substrate. Four chamber slides were coated with either Fn alone, Fn+galectin-1 (Gal-1), Lam alone, Lam+Gal-1, or Gal-1 alone. RPE cells were then plated at a density of 5×10 4 cells per chamber and allowed to spread. Results were obtained from evaluation of four separate fields by examining at least 100 cells per field. Values indicate means±SD of three experiments performed in duplicate and are expressed as percentage of cells spreading on Fn or Lam as a substrate. Statistical analysis was performed using Students t -test and a p value

    Article Snippet: After reaching confluency, primary RPE cells were subcultured and maintained in DMEM (Biochrom) supplemented with 10% FCS (Biochrom) at 37 °C and 5% CO2 .

    Techniques: Incubation, Laser Capture Microdissection, Staining

    Effect of OA-GL12 on the repair rate of HaCaT cells scratches HaCaT cells (2.5 × 10 5 /well) were cultured in 24-well plates for 12–24 h with serum starvation and then made to the mechanical scratch wound by a sterile pipette tip. After washed, cells were cultured for next periods (from 0 to 24 h) in a serum-free basal medium with the continued presence of OA-GL12. The same volume of DMEM (serum free) was added as vehicle and OM-LV20 (50 nM) as positive control. ( A ). OA-GL12 (10 pM) showed prominent wound-healing capacity on HaCaT cells. ( B ). OA-GL12 illustrated time- and concentration- dependent wound-healing capacity on HaCaT cells. Contain cells were added into 96-well plates with basic DMEM (90 μl) without 10% FBS and incubated for 2-4 h. Afterward, OA-GL12 (10 μl) was added to each well and incubated for 24 h at various final concentrations then tested using a CellTiter 96® AQueous One Solution Assay. ( C ). OM-LV20 (50 nM) showed proliferative effect, but OA-GL12 did not show on HaCaT cells from 2 to 10 pM. Data were presented as mean ± S.D., n =9. * P

    Journal: Bioscience Reports

    Article Title: A short peptide potentially promotes the healing of skin wound

    doi: 10.1042/BSR20181734

    Figure Lengend Snippet: Effect of OA-GL12 on the repair rate of HaCaT cells scratches HaCaT cells (2.5 × 10 5 /well) were cultured in 24-well plates for 12–24 h with serum starvation and then made to the mechanical scratch wound by a sterile pipette tip. After washed, cells were cultured for next periods (from 0 to 24 h) in a serum-free basal medium with the continued presence of OA-GL12. The same volume of DMEM (serum free) was added as vehicle and OM-LV20 (50 nM) as positive control. ( A ). OA-GL12 (10 pM) showed prominent wound-healing capacity on HaCaT cells. ( B ). OA-GL12 illustrated time- and concentration- dependent wound-healing capacity on HaCaT cells. Contain cells were added into 96-well plates with basic DMEM (90 μl) without 10% FBS and incubated for 2-4 h. Afterward, OA-GL12 (10 μl) was added to each well and incubated for 24 h at various final concentrations then tested using a CellTiter 96® AQueous One Solution Assay. ( C ). OM-LV20 (50 nM) showed proliferative effect, but OA-GL12 did not show on HaCaT cells from 2 to 10 pM. Data were presented as mean ± S.D., n =9. * P

    Article Snippet: Inflammatory assay RAW264.7 cells were cultured in DMEM (BI, Israel) supplemented with 10% (v/v) FBS Hyclone) and antibiotics (100 units/ml penicillin and 100 units/ml streptomycin) at 37°C in a humidified atmosphere of 5% CO2 .

    Techniques: Cell Culture, Transferring, Positive Control, Concentration Assay, Incubation

    Effect of OA-GL12 on the repair rate of HSF cell scratches HSF cells (2.5 × 10 5 /well) were cultured in 24-well plates for 12–24 h and then made to the mechanical scratch wound by a sterile pipette tip. After washing, cells were cultured for next periods in a serum-free basal medium (500 μl) with the continued presence of OA-GL12. The same volume of DMEM (serum free) was added as vehicle. ( A ) OA-GL12 (10 pM) illustrated distinct HSF cells scratch-healing activity. ( B ) OA-GL12 illustrated time- and concentration-dependent HSF cells scratch-healing activity. Contain cells were added into 96-well plates with basic DMEM (90 μl) without 10% FBS and incubated for 2–4 h. Afterward, OA-GL12 (10 μl) was added to each well and incubated for 24 h at various final concentrations then tested using a CellTiter 96® AQueous One Solution Assay. ( C , D ). OA-GL12 showed no impact on the proliferation of HSF and HUVEC cells from 2 pM to 10 nM. Data were presented as mean ± S.D., n =9. * P

    Journal: Bioscience Reports

    Article Title: A short peptide potentially promotes the healing of skin wound

    doi: 10.1042/BSR20181734

    Figure Lengend Snippet: Effect of OA-GL12 on the repair rate of HSF cell scratches HSF cells (2.5 × 10 5 /well) were cultured in 24-well plates for 12–24 h and then made to the mechanical scratch wound by a sterile pipette tip. After washing, cells were cultured for next periods in a serum-free basal medium (500 μl) with the continued presence of OA-GL12. The same volume of DMEM (serum free) was added as vehicle. ( A ) OA-GL12 (10 pM) illustrated distinct HSF cells scratch-healing activity. ( B ) OA-GL12 illustrated time- and concentration-dependent HSF cells scratch-healing activity. Contain cells were added into 96-well plates with basic DMEM (90 μl) without 10% FBS and incubated for 2–4 h. Afterward, OA-GL12 (10 μl) was added to each well and incubated for 24 h at various final concentrations then tested using a CellTiter 96® AQueous One Solution Assay. ( C , D ). OA-GL12 showed no impact on the proliferation of HSF and HUVEC cells from 2 pM to 10 nM. Data were presented as mean ± S.D., n =9. * P

    Article Snippet: Inflammatory assay RAW264.7 cells were cultured in DMEM (BI, Israel) supplemented with 10% (v/v) FBS Hyclone) and antibiotics (100 units/ml penicillin and 100 units/ml streptomycin) at 37°C in a humidified atmosphere of 5% CO2 .

    Techniques: Cell Culture, Transferring, Activity Assay, Concentration Assay, Incubation

    EA.hy926 cells secretome induce LOX-1 shedding. (A) Western blot analysis of conditioned medium derived from cross-cells experiments. LOX-1-V5 COS transfected cells were incubated for 10 min with DMEM (lane 1), medium derived from untreated EA cells (lane 2) and medium derived from EA cells conditioned in the presence of ox-LDL (20μg/ml) (lane 3). The histogram on the right shows the densitometric analysis of sLOX-1 band intensity. (B) Effect of different classes of protease inhibitors on LOX-1 shedding. Inhibitors were added during the 10 min final incubation time. GM6001 was used at 2.6 and 26μM (lanes 3 and 4), Pepstatin A at 10μM (lane 5) and PMSF at 2mM (lane 6).

    Journal: PLoS ONE

    Article Title: Membrane Cholesterol Modulates LOX-1 Shedding in Endothelial Cells

    doi: 10.1371/journal.pone.0141270

    Figure Lengend Snippet: EA.hy926 cells secretome induce LOX-1 shedding. (A) Western blot analysis of conditioned medium derived from cross-cells experiments. LOX-1-V5 COS transfected cells were incubated for 10 min with DMEM (lane 1), medium derived from untreated EA cells (lane 2) and medium derived from EA cells conditioned in the presence of ox-LDL (20μg/ml) (lane 3). The histogram on the right shows the densitometric analysis of sLOX-1 band intensity. (B) Effect of different classes of protease inhibitors on LOX-1 shedding. Inhibitors were added during the 10 min final incubation time. GM6001 was used at 2.6 and 26μM (lanes 3 and 4), Pepstatin A at 10μM (lane 5) and PMSF at 2mM (lane 6).

    Article Snippet: Cell cultures and transfection COS cells, HEK-293#19 [ ] and CHO-F2 clones stably expressing LOX-1-V5, and the human EA.hy926 endothelial cell line [ ] were grown in DMEM (Dulbecco’s modified Eagle’s medium) (Biowest, Miami, FL) supplemented with 10% fetal bovine serum (Gibco, Paisley, UK), L-glutamine 1mM (Sigma Aldrich, St. Louis, MO), sodium pyruvate 1mM (Biowest, Miami, FL) and 100U/ml penicillin-streptomycin (Euroclone, Devon, UK).

    Techniques: Western Blot, Derivative Assay, Transfection, Incubation

    Relationship between the incidence of PC and rate of proliferation in 184A1 and MDA-MB-231. ( A,B ) 184A1 was cultured in three different media: DMEM without FBS (D S−), DMEM with 10% FBS (D S+), and optimal basal medium with supplements and growth factors (MEM). PC were labeled by anti-acetylated α-tubulin (red) and proliferating cells by anti-Ki-67 (green). ( A ) PC were located only in non-proliferating Ki-67-negative cells. ( B ) The incidence of PC (red bars) in 184A1 cells was higher in serum-free conditions (D S−) and with longer duration of culture. Data are the mean and standard error of two independent experiments performed in triplicate. ( C ) MDA-MB-231 were grown in DMEM with (D S+) and without (D S−) serum. Proliferation (Ki-67 labeling, green bars) was lower in serum-free conditions; however, there was no corresponding statistically significant increase in PC (red bars). Data are the mean of two independent experiments performed in triplicate. Bar: A = 10 μm.

    Journal: Journal of Histochemistry and Cytochemistry

    Article Title: Primary Cilia Are Decreased in Breast Cancer: Analysis of a Collection of Human Breast Cancer Cell Lines and Tissues

    doi: 10.1369/jhc.2010.955856

    Figure Lengend Snippet: Relationship between the incidence of PC and rate of proliferation in 184A1 and MDA-MB-231. ( A,B ) 184A1 was cultured in three different media: DMEM without FBS (D S−), DMEM with 10% FBS (D S+), and optimal basal medium with supplements and growth factors (MEM). PC were labeled by anti-acetylated α-tubulin (red) and proliferating cells by anti-Ki-67 (green). ( A ) PC were located only in non-proliferating Ki-67-negative cells. ( B ) The incidence of PC (red bars) in 184A1 cells was higher in serum-free conditions (D S−) and with longer duration of culture. Data are the mean and standard error of two independent experiments performed in triplicate. ( C ) MDA-MB-231 were grown in DMEM with (D S+) and without (D S−) serum. Proliferation (Ki-67 labeling, green bars) was lower in serum-free conditions; however, there was no corresponding statistically significant increase in PC (red bars). Data are the mean of two independent experiments performed in triplicate. Bar: A = 10 μm.

    Article Snippet: MDA-MB-231, MDA-MB-361, MDA-MB-468, Hs578T, MDA-MB-435, MDA435/LCC6, MCF10DCIS.com, MCF10CA1, and MCF7 cells were maintained in DMEM (Cellgro; Manassas, VA) supplemented with 10% FBS (Hyclone; Logan, UT).

    Techniques: Multiple Displacement Amplification, Cell Culture, Labeling

    Effects of amino acids on ERK phosphorylation Panels A and B: Western blot demonstrating the time course of ERK phosphorylation elicited by meat extract a mixture of amino acids. BMSCs from C57BL/6 mice were grown in 60 mm 2 dishes in DMEM + 10% fetal calf serum to ~ 80% confluency, then switched to KRB buffer +0.2% BSA. After an overnight incubation, cells were stimulated with a 0.3% meat extract for the indicated times. Cellular protein was extracted and 50 μg/lane was separated by electrophoresis on SDS-PAGE gels. For Western blotting with ECL detection, antibodies directed against both total ERK and phospho-ERK were utilized. Primary (1:750, 4°C, overnight) and HRP-coupled secondary (1:2000, room temperature, 1 hour) antibodies were obtained from Cell Signaling Technology (Danvers, MA). Phorbol ester (TPA, 1 μM, 10 min) was used as a positive control. (A) Representative blot. (B) Quantitation by densitometric analysis of six different experiments from three different cell preparations. Values are means±SEMs. *P

    Journal: Bone

    Article Title: Amino Acids as Signaling Molecules Modulating Bone Turnover

    doi: 10.1016/j.bone.2018.02.028

    Figure Lengend Snippet: Effects of amino acids on ERK phosphorylation Panels A and B: Western blot demonstrating the time course of ERK phosphorylation elicited by meat extract a mixture of amino acids. BMSCs from C57BL/6 mice were grown in 60 mm 2 dishes in DMEM + 10% fetal calf serum to ~ 80% confluency, then switched to KRB buffer +0.2% BSA. After an overnight incubation, cells were stimulated with a 0.3% meat extract for the indicated times. Cellular protein was extracted and 50 μg/lane was separated by electrophoresis on SDS-PAGE gels. For Western blotting with ECL detection, antibodies directed against both total ERK and phospho-ERK were utilized. Primary (1:750, 4°C, overnight) and HRP-coupled secondary (1:2000, room temperature, 1 hour) antibodies were obtained from Cell Signaling Technology (Danvers, MA). Phorbol ester (TPA, 1 μM, 10 min) was used as a positive control. (A) Representative blot. (B) Quantitation by densitometric analysis of six different experiments from three different cell preparations. Values are means±SEMs. *P

    Article Snippet: BMSCs were cultured in either DMEM (Corning Cellgro, low glucose #10-014CV) or M199 (Corning Cellgro # 10-060-CV).

    Techniques: Western Blot, Mouse Assay, Incubation, Electrophoresis, SDS Page, Positive Control, Quantitation Assay

    SCD1 is needed for MCF-7 and MDA-MB-231 cell migration. ( A ) Pharmacological inactivation of SCD1 with the small-molecule inhibitor A939572 decreased the migratory ability of both MCF-7 and MDA-MB-231 cells in an in vitro wound-healing assay. Cells were treated with 1 μ M A939572 or with vehicle (control, ctrl) in serum-free DMEM for 24–48 h prior to assay. ( B ) siRNA-mediated knockdown of SCD1 reduced MCF-7 and MDA-MB-231 migration in an in vitro wound-healing assay. Cell were transiently transfected for 72 h with 60 pmol of either siRNA ctrl and siRNA SCD1 prior to assay. ( A , B ) Cell proliferation was prevented by a 2 h pretreatment with mitomycin C (5 μ g ml −1 ). A vertical scratch was made with a pipette tip in the confluent cell monolayers and cell motility assayed at the indicated times ( x -axis). The gaps were photographed at 0, 24 and 48 h after wounding (original magnification × 100) and the distance migrated was measured in micrometres ( y -axis) relative to zero time using ImageJ software. All experiments were run in triplicate and repeated three times. The data shown are the mean±s.e. * P

    Journal: British Journal of Cancer

    Article Title: Stearoyl-CoA desaturase 1 and paracrine diffusible signals have a major role in the promotion of breast cancer cell migration induced by cancer-associated fibroblasts

    doi: 10.1038/bjc.2015.135

    Figure Lengend Snippet: SCD1 is needed for MCF-7 and MDA-MB-231 cell migration. ( A ) Pharmacological inactivation of SCD1 with the small-molecule inhibitor A939572 decreased the migratory ability of both MCF-7 and MDA-MB-231 cells in an in vitro wound-healing assay. Cells were treated with 1 μ M A939572 or with vehicle (control, ctrl) in serum-free DMEM for 24–48 h prior to assay. ( B ) siRNA-mediated knockdown of SCD1 reduced MCF-7 and MDA-MB-231 migration in an in vitro wound-healing assay. Cell were transiently transfected for 72 h with 60 pmol of either siRNA ctrl and siRNA SCD1 prior to assay. ( A , B ) Cell proliferation was prevented by a 2 h pretreatment with mitomycin C (5 μ g ml −1 ). A vertical scratch was made with a pipette tip in the confluent cell monolayers and cell motility assayed at the indicated times ( x -axis). The gaps were photographed at 0, 24 and 48 h after wounding (original magnification × 100) and the distance migrated was measured in micrometres ( y -axis) relative to zero time using ImageJ software. All experiments were run in triplicate and repeated three times. The data shown are the mean±s.e. * P

    Article Snippet: MCF-7 and MDA-MB-231 cells were maintained in DMEM (Euroclone) supplemented with 10% FBS (Life Technologies, Paisley, Scotland), antibiotics (100 IU ml−1 penicillin, 100 μ g ml−1 streptomycin, Euroclone) and 2 mM glutamine (Euroclone).

    Techniques: Multiple Displacement Amplification, Migration, In Vitro, Wound Healing Assay, Transfection, Transferring, Software

    The LRS of SQSTM1 and TBC1D25 is essential for phagophore localization, but not sufficient for their degradation. (A) Schematic representation of the LRS-swapping mutants of TBC1D25 and SQSTM1, named TBC1D25-(SQT-LRS) and SQSTM1-(TBC-LRS), respectively. The asterisks indicate the residues that are highly conserved in many LRSs. The WA mutant contains a Trp-to-Ala (WA) mutation (shaded background), which disrupts binding activity to LC3 homologs. (B) Differing LC3 homolog binding activity of TBC1D25, TBC1D25-W136A (designated as WA), and TBC1D25-(SQT-LRS) (designated as SQT) as revealed by GST affinity isolation assays. COS-7 cell lysates expressing EGFP-TBC1D25, EGFP-TBC1D25-WA, or EGFP-TBC1D25-(SQT-LRS) were incubated with GST-LC3, GST-GABARAP, or GST-GABARAPL2 that had been coupled with glutathione-Sepharose beads. Proteins bound to the beads were analyzed by immunoblotting with HRP-conjugated anti-GFP antibody (top panel) and HRP-conjugated anti-GST antibody (bottom panel). The asterisks indicate the nonspecific bands of the anti-GFP antibody. (C) sqstm1 -KO MEF cells stably expressing FLAG-SQSTM1, FLAG-SQSTM1-WA, FLAG-SQSTM1-(TBC-LRS) (FLAG-SQSTM1; left panels), FLAG-TBC1D25, FLAG-TBC1D25-WA, or FLAG-TBC1D25-(SQT-LRS) (FLAG-TBC1D25; right panels) were cultured for 2 h in DMEM (nutrient-rich; - starvation) or HBSS (starvation) with or without 20 μM BafA1. Cell lysates were analyzed by immunoblotting with anti-FLAG tag antibody (top panels) and anti-ACTB antibody (bottom panels). The positions of the molecular mass markers (in kilodaltons) are shown on the left.

    Journal: Autophagy

    Article Title: Differing susceptibility to autophagic degradation of two LC3-binding proteins: SQSTM1/p62 and TBC1D25/OATL1

    doi: 10.1080/15548627.2015.1124223

    Figure Lengend Snippet: The LRS of SQSTM1 and TBC1D25 is essential for phagophore localization, but not sufficient for their degradation. (A) Schematic representation of the LRS-swapping mutants of TBC1D25 and SQSTM1, named TBC1D25-(SQT-LRS) and SQSTM1-(TBC-LRS), respectively. The asterisks indicate the residues that are highly conserved in many LRSs. The WA mutant contains a Trp-to-Ala (WA) mutation (shaded background), which disrupts binding activity to LC3 homologs. (B) Differing LC3 homolog binding activity of TBC1D25, TBC1D25-W136A (designated as WA), and TBC1D25-(SQT-LRS) (designated as SQT) as revealed by GST affinity isolation assays. COS-7 cell lysates expressing EGFP-TBC1D25, EGFP-TBC1D25-WA, or EGFP-TBC1D25-(SQT-LRS) were incubated with GST-LC3, GST-GABARAP, or GST-GABARAPL2 that had been coupled with glutathione-Sepharose beads. Proteins bound to the beads were analyzed by immunoblotting with HRP-conjugated anti-GFP antibody (top panel) and HRP-conjugated anti-GST antibody (bottom panel). The asterisks indicate the nonspecific bands of the anti-GFP antibody. (C) sqstm1 -KO MEF cells stably expressing FLAG-SQSTM1, FLAG-SQSTM1-WA, FLAG-SQSTM1-(TBC-LRS) (FLAG-SQSTM1; left panels), FLAG-TBC1D25, FLAG-TBC1D25-WA, or FLAG-TBC1D25-(SQT-LRS) (FLAG-TBC1D25; right panels) were cultured for 2 h in DMEM (nutrient-rich; - starvation) or HBSS (starvation) with or without 20 μM BafA1. Cell lysates were analyzed by immunoblotting with anti-FLAG tag antibody (top panels) and anti-ACTB antibody (bottom panels). The positions of the molecular mass markers (in kilodaltons) are shown on the left.

    Article Snippet: MEF cells and COS-7 cells were maintained at 37°C in DMEM (Wako Pure Chemical Industries, Ltd., 044-29765) containing 10% fetal bovine serum and the antibiotics streptomycin and penicillin-G under a 5% CO2 atmosphere.

    Techniques: Mutagenesis, Binding Assay, Activity Assay, Isolation, Expressing, Incubation, Stable Transfection, Cell Culture, FLAG-tag

    Colocalization of TBC1D25-chimera mutants with LC3. sqstm1 -KO MEF cells stably expressing EGFP-TBC1D25ΔN, EGFP-TBC1D25ΔN-WA, EGFP-PB1-TBC1D25ΔN, EGFP-PB1-TBC1D25ΔN-WA, EGFP-TBC1D25ΔN-UBA, EGFP-TBC1D25ΔN-WA-UBA, EGFP-PB1-TBC1D25ΔN-UBA, or EGFP-PB1-TBC1D25ΔN-WA-UBA were cultured for 1 h in DMEM (nutrient-rich; far left column) or HBSS (starvation; right 3 columns). Note that cytoplasmic dots were often observed in the cells expressing an TBC1D25 mutant with the LRS that contained a PB1 domain, i.e., EGFP-PB1-TBC1D25ΔN and EGFP-PB1-TBC1D25ΔN-UBA, even under nutrient-rich conditions, whereas no dots were observed with other TBC1D25 mutants under nutrient-rich conditions. Scale bars: 10 μm.

    Journal: Autophagy

    Article Title: Differing susceptibility to autophagic degradation of two LC3-binding proteins: SQSTM1/p62 and TBC1D25/OATL1

    doi: 10.1080/15548627.2015.1124223

    Figure Lengend Snippet: Colocalization of TBC1D25-chimera mutants with LC3. sqstm1 -KO MEF cells stably expressing EGFP-TBC1D25ΔN, EGFP-TBC1D25ΔN-WA, EGFP-PB1-TBC1D25ΔN, EGFP-PB1-TBC1D25ΔN-WA, EGFP-TBC1D25ΔN-UBA, EGFP-TBC1D25ΔN-WA-UBA, EGFP-PB1-TBC1D25ΔN-UBA, or EGFP-PB1-TBC1D25ΔN-WA-UBA were cultured for 1 h in DMEM (nutrient-rich; far left column) or HBSS (starvation; right 3 columns). Note that cytoplasmic dots were often observed in the cells expressing an TBC1D25 mutant with the LRS that contained a PB1 domain, i.e., EGFP-PB1-TBC1D25ΔN and EGFP-PB1-TBC1D25ΔN-UBA, even under nutrient-rich conditions, whereas no dots were observed with other TBC1D25 mutants under nutrient-rich conditions. Scale bars: 10 μm.

    Article Snippet: MEF cells and COS-7 cells were maintained at 37°C in DMEM (Wako Pure Chemical Industries, Ltd., 044-29765) containing 10% fetal bovine serum and the antibiotics streptomycin and penicillin-G under a 5% CO2 atmosphere.

    Techniques: Stable Transfection, Expressing, Cell Culture, Mutagenesis

    TBC1D25 is not a substrate of starvation-induced autophagy. (A) Domain organization of TBC1D25 and sequestosome-1 (SQSTM1) and sequence alignment of the PB1-like domain of TBC1D25 and PB1 domain of SQSTM1. The N-terminal domains of TBC1D25 and SQSTM1 were aligned by using the HHpred software program (see also Fig. S2). Amino acid residues in the sequences that are identical and similar are shown against a black background and a shaded background, respectively. (B) MEF cells stably expressing EGFP-TBC1D25 were cultured for 1 h under nutrient-rich conditions (far left column) or starved conditions (right 3 columns). Note that EGFP-TBC1D25 and endogenous SQSTM1 colocalized well with LC3 dots under starved conditions. Scale bars: 10 μm. (C) The TBC1D25 protein level in both control MEF cells and sqstm1 -KO MEF cells was unaltered by starvation. Control MEF cells and sqstm1 -KO MEF cells were cultured for 2 h in DMEM (nutrient-rich; - starvation) or HBSS (starvation) with or without 100 μM E64d and 100 μg/ml pepstatin A, and their cell lysates were analyzed by immunoblotting with anti-TBC1D25 antibody (top panel), anti-SQSTM1 antibody (middle panel), and anti-ACTB antibody (bottom panel). Note that SQSTM1, but not TBC1D25, was degraded by starvation. The asterisk indicates the nonspecific band of the anti-SQSTM1 antibody. The positions of the molecular mass markers (in kilodaltons) are shown on the left.

    Journal: Autophagy

    Article Title: Differing susceptibility to autophagic degradation of two LC3-binding proteins: SQSTM1/p62 and TBC1D25/OATL1

    doi: 10.1080/15548627.2015.1124223

    Figure Lengend Snippet: TBC1D25 is not a substrate of starvation-induced autophagy. (A) Domain organization of TBC1D25 and sequestosome-1 (SQSTM1) and sequence alignment of the PB1-like domain of TBC1D25 and PB1 domain of SQSTM1. The N-terminal domains of TBC1D25 and SQSTM1 were aligned by using the HHpred software program (see also Fig. S2). Amino acid residues in the sequences that are identical and similar are shown against a black background and a shaded background, respectively. (B) MEF cells stably expressing EGFP-TBC1D25 were cultured for 1 h under nutrient-rich conditions (far left column) or starved conditions (right 3 columns). Note that EGFP-TBC1D25 and endogenous SQSTM1 colocalized well with LC3 dots under starved conditions. Scale bars: 10 μm. (C) The TBC1D25 protein level in both control MEF cells and sqstm1 -KO MEF cells was unaltered by starvation. Control MEF cells and sqstm1 -KO MEF cells were cultured for 2 h in DMEM (nutrient-rich; - starvation) or HBSS (starvation) with or without 100 μM E64d and 100 μg/ml pepstatin A, and their cell lysates were analyzed by immunoblotting with anti-TBC1D25 antibody (top panel), anti-SQSTM1 antibody (middle panel), and anti-ACTB antibody (bottom panel). Note that SQSTM1, but not TBC1D25, was degraded by starvation. The asterisk indicates the nonspecific band of the anti-SQSTM1 antibody. The positions of the molecular mass markers (in kilodaltons) are shown on the left.

    Article Snippet: MEF cells and COS-7 cells were maintained at 37°C in DMEM (Wako Pure Chemical Industries, Ltd., 044-29765) containing 10% fetal bovine serum and the antibiotics streptomycin and penicillin-G under a 5% CO2 atmosphere.

    Techniques: Sequencing, Software, Stable Transfection, Expressing, Cell Culture

    The PB1 domain of SQSTM1 is necessary for starvation-induced degradation of TBC1D25-chimera mutants. (A) sqstm1 -KO MEF cells stably expressing the indicated FLAG-tagged SQSTM1 (wild-type and WA) and TBC1D25 chimera mutants were cultured for 2 h in DMEM (nutrient-rich; - starvation) or HBSS (starvation). Cell lysates were analyzed by immunoblotting with anti-FLAG tag antibody (top panel) and anti-ACTB antibody (bottom panel). The levels of PB1-TBC1D25ΔN expression (lanes 9 and 10) and PB1-TBC1D25ΔN-UBA expression (lanes 17 and 18) were much lower than the levels of expression of other TBC1D25 mutants, because both of these proteins were degraded by basal autophagy even under nutrient-rich conditions (see Fig. S3B). (B) Quantification of the band intensity of FLAG-tagged proteins shown in the top panel in A . The bars represent the means and SE of data from 3 independent experiments. **, P

    Journal: Autophagy

    Article Title: Differing susceptibility to autophagic degradation of two LC3-binding proteins: SQSTM1/p62 and TBC1D25/OATL1

    doi: 10.1080/15548627.2015.1124223

    Figure Lengend Snippet: The PB1 domain of SQSTM1 is necessary for starvation-induced degradation of TBC1D25-chimera mutants. (A) sqstm1 -KO MEF cells stably expressing the indicated FLAG-tagged SQSTM1 (wild-type and WA) and TBC1D25 chimera mutants were cultured for 2 h in DMEM (nutrient-rich; - starvation) or HBSS (starvation). Cell lysates were analyzed by immunoblotting with anti-FLAG tag antibody (top panel) and anti-ACTB antibody (bottom panel). The levels of PB1-TBC1D25ΔN expression (lanes 9 and 10) and PB1-TBC1D25ΔN-UBA expression (lanes 17 and 18) were much lower than the levels of expression of other TBC1D25 mutants, because both of these proteins were degraded by basal autophagy even under nutrient-rich conditions (see Fig. S3B). (B) Quantification of the band intensity of FLAG-tagged proteins shown in the top panel in A . The bars represent the means and SE of data from 3 independent experiments. **, P

    Article Snippet: MEF cells and COS-7 cells were maintained at 37°C in DMEM (Wako Pure Chemical Industries, Ltd., 044-29765) containing 10% fetal bovine serum and the antibiotics streptomycin and penicillin-G under a 5% CO2 atmosphere.

    Techniques: Stable Transfection, Expressing, Cell Culture, FLAG-tag

    Metabolic inhibition of sulfation of HS chains leads to a loss of sulfated groups of HS, Hsp90α, and Hsp90β from the cell surface ( a ) and to an inhibition of the basal and Hsp90-stimulated cell migration and invasion ( b – d ). a Cells were treated with sodium chlorate, after which cells were grown at 37 °C in DMEM-FBS without chlorate. At indicated times, cell surface HS and proteins were stained with specific antibodies, analyzed by flow cytometry, and quantified. The data are presented as the MFI specific for HS, Hsp90α, Hsp90β, and LRP1, expressed in percent. The specific MFI of control untreated cells was taken as 100%. b The migration of chlorate-treated cells in the wound-healing assay in the presence and absence of Hsp90. c The analysis of the basal migration/invasion of chlorate-treated cells. The basal migration/invasion of cells expressed in percent is presented. The basal migration/invasion of untreated control cells was taken as 100%. d ” section and expressed in percent. The Hsp90-stimulated migration/invasion of control untreated cells was taken as 100%. a , c , d Each bar represents the mean ± SD ( n = 3–4). * p

    Journal: Cell Stress & Chaperones

    Article Title: Cell surface heparan sulfate proteoglycans are involved in the extracellular Hsp90-stimulated migration and invasion of cancer cells

    doi: 10.1007/s12192-018-0955-5

    Figure Lengend Snippet: Metabolic inhibition of sulfation of HS chains leads to a loss of sulfated groups of HS, Hsp90α, and Hsp90β from the cell surface ( a ) and to an inhibition of the basal and Hsp90-stimulated cell migration and invasion ( b – d ). a Cells were treated with sodium chlorate, after which cells were grown at 37 °C in DMEM-FBS without chlorate. At indicated times, cell surface HS and proteins were stained with specific antibodies, analyzed by flow cytometry, and quantified. The data are presented as the MFI specific for HS, Hsp90α, Hsp90β, and LRP1, expressed in percent. The specific MFI of control untreated cells was taken as 100%. b The migration of chlorate-treated cells in the wound-healing assay in the presence and absence of Hsp90. c The analysis of the basal migration/invasion of chlorate-treated cells. The basal migration/invasion of cells expressed in percent is presented. The basal migration/invasion of untreated control cells was taken as 100%. d ” section and expressed in percent. The Hsp90-stimulated migration/invasion of control untreated cells was taken as 100%. a , c , d Each bar represents the mean ± SD ( n = 3–4). * p

    Article Snippet: FBS and DMEM were purchased from GE Healthcare.

    Techniques: Inhibition, Migration, Staining, Flow Cytometry, Cytometry, Wound Healing Assay

    Digestion of HS moieties of HSPGs with a heparinase reduces the level of HS and Hsp90s at the cell surface ( a ) and inhibits the basal and Hsp90-stimulated cell migration and invasion ( b – d ). a Cells were treated with a heparinase, after which cells were grown at 37 °C in DMEM-FBS. At indicated times, cell surface proteins were stained with specific antibodies, analyzed by flow cytometry, and quantified. The data are presented as the MFI specific for HS, Hsp90α, Hsp90β, and LRP1, expressed in percent. The specific MFI of control untreated cells was taken as 100%. b The migration of heparinase-treated cells in the wound-healing assay in the presence and absence of Hsp90. c The analysis of the basal migration/invasion of heparinase-treated cells. The migration/invasion of cells expressed in percent is presented. The basal migration/invasion of untreated control cells was taken as 100%. d ” section and expressed in percent. The Hsp90-stimulated migration/invasion of control untreated cells was taken as 100%. a , c , d Each bar represents the mean ± SD ( n = 3–4). * p

    Journal: Cell Stress & Chaperones

    Article Title: Cell surface heparan sulfate proteoglycans are involved in the extracellular Hsp90-stimulated migration and invasion of cancer cells

    doi: 10.1007/s12192-018-0955-5

    Figure Lengend Snippet: Digestion of HS moieties of HSPGs with a heparinase reduces the level of HS and Hsp90s at the cell surface ( a ) and inhibits the basal and Hsp90-stimulated cell migration and invasion ( b – d ). a Cells were treated with a heparinase, after which cells were grown at 37 °C in DMEM-FBS. At indicated times, cell surface proteins were stained with specific antibodies, analyzed by flow cytometry, and quantified. The data are presented as the MFI specific for HS, Hsp90α, Hsp90β, and LRP1, expressed in percent. The specific MFI of control untreated cells was taken as 100%. b The migration of heparinase-treated cells in the wound-healing assay in the presence and absence of Hsp90. c The analysis of the basal migration/invasion of heparinase-treated cells. The migration/invasion of cells expressed in percent is presented. The basal migration/invasion of untreated control cells was taken as 100%. d ” section and expressed in percent. The Hsp90-stimulated migration/invasion of control untreated cells was taken as 100%. a , c , d Each bar represents the mean ± SD ( n = 3–4). * p

    Article Snippet: FBS and DMEM were purchased from GE Healthcare.

    Techniques: Migration, Staining, Flow Cytometry, Cytometry, Wound Healing Assay

    Haemocytometer and Microscopy based HeLa Cell Proliferation Data is Presented. ( Figure 2A ) represents the photomicrograph taken at 10X of HeLa growing in six well plate with different treatment condition whereas DMEM Control, EFM: DMEM and BCEF: DMEM. The Microscopy photographs were taken at the end of treatment at 48 h. The Microscopy photographs were captured using light inverted microscope. ( Figure 2B ) is a graphical representative oftotal cells after treatment condition (DMEM Control DMEM Control, EFM: DMEM and BCEF: DMEM). The Total cells were recorded using Haemocytometer and data is presented as Percentage of Total Cell Count X 10 4 in each treatment conditions. The Data are represented as Mean ± SD. Each experiment was conducted independently four times. ( Figure 2C ) shows us the percent viability of cells after the respective treatment conditions. ( Figure 2D ) The photograph depicts the photomicrograph taken at 10X for HeLa cell growing in 96 Well Plate with different treatment condition (DMEM Control, EFM: DMEM and BCEF: DMEM). The microscopy photographs were taken at the end of MTT based cytotoxicity assay showing Formazan crystals formed, using 10 X Objective. ( Figure 2E ) Graphical representation of absorbance captured by ELISA plate reader with cells treated with DMEM Control DMEM Control, EFM: DMEM and BCEF: DMEM. Total cells were recorded by taking absorbance with ELISA reader. The Data are represented as Mean ± SD. Each experiment was conducted independently three times.

    Journal: Asian Pacific Journal of Cancer Prevention : APJCP

    Article Title: Induction of Apoptotic Death and Cell Cycle Arrest in HeLa Cells by Extracellular Factors of Breast Cancer Cells

    doi: 10.31557/APJCP.2018.19.12.3307

    Figure Lengend Snippet: Haemocytometer and Microscopy based HeLa Cell Proliferation Data is Presented. ( Figure 2A ) represents the photomicrograph taken at 10X of HeLa growing in six well plate with different treatment condition whereas DMEM Control, EFM: DMEM and BCEF: DMEM. The Microscopy photographs were taken at the end of treatment at 48 h. The Microscopy photographs were captured using light inverted microscope. ( Figure 2B ) is a graphical representative oftotal cells after treatment condition (DMEM Control DMEM Control, EFM: DMEM and BCEF: DMEM). The Total cells were recorded using Haemocytometer and data is presented as Percentage of Total Cell Count X 10 4 in each treatment conditions. The Data are represented as Mean ± SD. Each experiment was conducted independently four times. ( Figure 2C ) shows us the percent viability of cells after the respective treatment conditions. ( Figure 2D ) The photograph depicts the photomicrograph taken at 10X for HeLa cell growing in 96 Well Plate with different treatment condition (DMEM Control, EFM: DMEM and BCEF: DMEM). The microscopy photographs were taken at the end of MTT based cytotoxicity assay showing Formazan crystals formed, using 10 X Objective. ( Figure 2E ) Graphical representation of absorbance captured by ELISA plate reader with cells treated with DMEM Control DMEM Control, EFM: DMEM and BCEF: DMEM. Total cells were recorded by taking absorbance with ELISA reader. The Data are represented as Mean ± SD. Each experiment was conducted independently three times.

    Article Snippet: Cell line maintenance and Seeding HeLa cells were cultured and maintained in DMEM (Dulbecco’s Modified Eagles Medium) (Himedia) with high glucose at 37°C and supplemented with 10% NBCS (New Born Calf Serum) (Himedia) and penicillin and streptomycin 100µg/ml.

    Techniques: Microscopy, Inverted Microscopy, Cell Counting, MTT Assay, Cytotoxicity Assay, Enzyme-linked Immunosorbent Assay

    THS extracts made in basal DMEM without serum proteins showed little cytotoxicity to mNSC. (A–D) Extracts were prepared from terry cloth exposed to cigarette smoke for different times and tested with mNSC in the MTT assay. (E) Dose response curves showing the cytotoxicity of THS extracts prepared from terry cloth exposed to 54 cigarettes over 8 months with and without serum proteins. Each curve represents mean ± SEM of three experiments. * = p

    Journal: Toxicology in vitro : an international journal published in association with BIBRA

    Article Title: Thirdhand smoke: Chemical dynamics, cytotoxicity, and genotoxicity in outdoor and indoor environments

    doi: 10.1016/j.tiv.2015.12.007

    Figure Lengend Snippet: THS extracts made in basal DMEM without serum proteins showed little cytotoxicity to mNSC. (A–D) Extracts were prepared from terry cloth exposed to cigarette smoke for different times and tested with mNSC in the MTT assay. (E) Dose response curves showing the cytotoxicity of THS extracts prepared from terry cloth exposed to 54 cigarettes over 8 months with and without serum proteins. Each curve represents mean ± SEM of three experiments. * = p

    Article Snippet: mNSC were cultured in DMEM (Lonza, Walkersville, MD) containing 10% fetal bovine serum, 5% horse serum, 1% sodium pyruvate (Lonza, Walkersville, MD) and 1% penicillin-streptomycin (GIBCO, Invitrogen, Carlsbad, CA) ( , ).

    Techniques: MTT Assay

    Effect of IBTP treatment on mitochondrial respiration of MB231 cells. Panel A: Cells plated on XF24 plates were treated with the indicated concentrations of IBTP or BTPP for 4h in 0.5% FBS-containing medium. After treatment, the medium was removed and replaced with XF assay medium (DMEM, containing 5mM glucose, 0.5% FBS, 5mM HEPES without bicarbonate) and equilibrated 1h before OCR measurement. Panel B: Cells plated on 6-well plates were treated with the indicated concentrations of IBTP or BTPP for 4h. After the incubation, the cells were harvested immediately by trypsinization. The harvested cells were replated in XF24 plates and allowed adhere for an additional 20h in complete medium containing 10% FBS (total 24h). The medium was removed and replaced with assay medium and equilibrated 1h before OCR measurement. Panel C : After 4h of IBTP or BTPP treatment, the medium was replaced with complete medium containing 10% FBS, and incubated for 48h. The cells were harvested after 48h, replated in XF24 plates and allowed adhere for an additional 20h in complete medium. The medium was replaced with assay media and incubated 1h before measurement of OCR (total duration 72h).

    Journal: PLoS ONE

    Article Title: A Novel Class of Mitochondria-Targeted Soft Electrophiles Modifies Mitochondrial Proteins and Inhibits Mitochondrial Metabolism in Breast Cancer Cells through Redox Mechanisms

    doi: 10.1371/journal.pone.0120460

    Figure Lengend Snippet: Effect of IBTP treatment on mitochondrial respiration of MB231 cells. Panel A: Cells plated on XF24 plates were treated with the indicated concentrations of IBTP or BTPP for 4h in 0.5% FBS-containing medium. After treatment, the medium was removed and replaced with XF assay medium (DMEM, containing 5mM glucose, 0.5% FBS, 5mM HEPES without bicarbonate) and equilibrated 1h before OCR measurement. Panel B: Cells plated on 6-well plates were treated with the indicated concentrations of IBTP or BTPP for 4h. After the incubation, the cells were harvested immediately by trypsinization. The harvested cells were replated in XF24 plates and allowed adhere for an additional 20h in complete medium containing 10% FBS (total 24h). The medium was removed and replaced with assay medium and equilibrated 1h before OCR measurement. Panel C : After 4h of IBTP or BTPP treatment, the medium was replaced with complete medium containing 10% FBS, and incubated for 48h. The cells were harvested after 48h, replated in XF24 plates and allowed adhere for an additional 20h in complete medium. The medium was replaced with assay media and incubated 1h before measurement of OCR (total duration 72h).

    Article Snippet: Breast cancer cell lines MDA-MB-231 (MB231) and MDA-MB-468 (MB468) human breast adenocarcinoma cells were cultured in DMEM (Mediatech, Manassas, VA) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals, Atlanta, GA).

    Techniques: XF Assay, Incubation

    Sequence comparison of ECs1581 variants in E. coli . A. Multiple alignment of the predicted amino acid sequences of ECs1581 in different lineages of O157:H7. Residue divergence specific to lineage I EHEC O157:H7 isolates is marked by a pink shaded box. B. Multiple alignment of EHEC variant ECs1581 (EDL933/Sakai) with commensal variant ECED1_1787 (ED1a) and UPEC variant C1493 (CFT073). The RGD motif is at amino acid position 20–22. Residue divergence between the strains is indicated. C. SDS-PAGE gel showing stimulation of T3S in an EHEC Δ ecs1581 mutant expressing EHEC ECs1581 (pECs1581) or UPEC C1493 (pC1493) from a low copy number plasmid. pWControl alone in the ecs1581 mutant was used as a negative control strain. TCA-precipitated culture supernatants were analysed as described previously in Experimental procedures . D. Analysis of motility repression in an EHEC Δ ecs1581 and UPEC Δ c1493 deletion background expressing pECs1581, pC1493 or pWControl. Motility was assessed as described in Experimental procedures . E. SDS-PAGE gel showing T3S profile for EHEC strain ZAP193 and isogenic Δ grlA ::Tn mutant expressing pECs1581 or pWControl. Strains were cultured in DMEM to an OD 600 of 1.0 and culture supernatant proteins were analysed exactly as described for strains grown in MEM-HEPES.

    Journal: Molecular Microbiology

    Article Title: Identification of a novel prophage regulator in Escherichia coli controlling the expression of type III secretion

    doi: 10.1111/j.1365-2958.2011.07927.x

    Figure Lengend Snippet: Sequence comparison of ECs1581 variants in E. coli . A. Multiple alignment of the predicted amino acid sequences of ECs1581 in different lineages of O157:H7. Residue divergence specific to lineage I EHEC O157:H7 isolates is marked by a pink shaded box. B. Multiple alignment of EHEC variant ECs1581 (EDL933/Sakai) with commensal variant ECED1_1787 (ED1a) and UPEC variant C1493 (CFT073). The RGD motif is at amino acid position 20–22. Residue divergence between the strains is indicated. C. SDS-PAGE gel showing stimulation of T3S in an EHEC Δ ecs1581 mutant expressing EHEC ECs1581 (pECs1581) or UPEC C1493 (pC1493) from a low copy number plasmid. pWControl alone in the ecs1581 mutant was used as a negative control strain. TCA-precipitated culture supernatants were analysed as described previously in Experimental procedures . D. Analysis of motility repression in an EHEC Δ ecs1581 and UPEC Δ c1493 deletion background expressing pECs1581, pC1493 or pWControl. Motility was assessed as described in Experimental procedures . E. SDS-PAGE gel showing T3S profile for EHEC strain ZAP193 and isogenic Δ grlA ::Tn mutant expressing pECs1581 or pWControl. Strains were cultured in DMEM to an OD 600 of 1.0 and culture supernatant proteins were analysed exactly as described for strains grown in MEM-HEPES.

    Article Snippet: In the analysis of T3S proteins, either DMEM (Sigma-Aldrich) or MEM-HEPES (Sigma-Aldrich) supplemented with 250 nM Fe(NO3 )2 and 0.2% glucose was used for culturing strains.

    Techniques: Sequencing, Variant Assay, SDS Page, Mutagenesis, Expressing, Low Copy Number, Plasmid Preparation, Negative Control, Cell Culture

    ECs1581 stimulates T3S via LEE1. A. Measurement of LEE1 promoter activity in EHEC strain TUV93-0 and isogenic Δ ecs1581 mutant. A 428 bp LEE1 promoter fragment (−444 to −16 upstream from the ler ATG start codon) was cloned into the promoter-less green fluorescence protein (GFP) plasmid pKC26 to create transcriptional fusion plasmid pLEE1–GFP. Strains transformed with this reporter were cultured in DMEM and the fluorescence produced by each bacterial population measured every 60 min as described in the Experimental procedures . Corresponding OD 600 measurements were taken at each time point and plotted against the mean fluorescence intensity values for each strain. The promoter-less plasmid pKC26 in each strain background acted as a control. B. Measurement of the pLEE1–GFP reporter and control plasmids in EHEC strain ZAP198 ( Table S1 ) with and without induced expression of ecs1581 (pECs1581) from low copy number plasmid pWSK29. C. Measurement of the pLEE1–GFP reporter and control plasmids in strain ZAP198 Δler (ZAP1004, Table S1 ) with and without induced expression of pECs1581. D. Measurement of the pLEE1–GFP reporter and control plasmids in strain ZAP198 with a constitutive LEE1 promoter (ZAP1327, Table S1 ) with and without induced expression of pECs1581. E. RT-PCR measurement of ler expression levels in EHEC strains ZAP198, ZAP1004 and ZAP1327. F. Western blot of EspD secretion by EHEC Δ ecs1581 and the replaced LEE1 promoter mutant (ZAP1327) trans expressing Ler (pLer) or ECs1581 (pECs1581) from low copy number plasmids.

    Journal: Molecular Microbiology

    Article Title: Identification of a novel prophage regulator in Escherichia coli controlling the expression of type III secretion

    doi: 10.1111/j.1365-2958.2011.07927.x

    Figure Lengend Snippet: ECs1581 stimulates T3S via LEE1. A. Measurement of LEE1 promoter activity in EHEC strain TUV93-0 and isogenic Δ ecs1581 mutant. A 428 bp LEE1 promoter fragment (−444 to −16 upstream from the ler ATG start codon) was cloned into the promoter-less green fluorescence protein (GFP) plasmid pKC26 to create transcriptional fusion plasmid pLEE1–GFP. Strains transformed with this reporter were cultured in DMEM and the fluorescence produced by each bacterial population measured every 60 min as described in the Experimental procedures . Corresponding OD 600 measurements were taken at each time point and plotted against the mean fluorescence intensity values for each strain. The promoter-less plasmid pKC26 in each strain background acted as a control. B. Measurement of the pLEE1–GFP reporter and control plasmids in EHEC strain ZAP198 ( Table S1 ) with and without induced expression of ecs1581 (pECs1581) from low copy number plasmid pWSK29. C. Measurement of the pLEE1–GFP reporter and control plasmids in strain ZAP198 Δler (ZAP1004, Table S1 ) with and without induced expression of pECs1581. D. Measurement of the pLEE1–GFP reporter and control plasmids in strain ZAP198 with a constitutive LEE1 promoter (ZAP1327, Table S1 ) with and without induced expression of pECs1581. E. RT-PCR measurement of ler expression levels in EHEC strains ZAP198, ZAP1004 and ZAP1327. F. Western blot of EspD secretion by EHEC Δ ecs1581 and the replaced LEE1 promoter mutant (ZAP1327) trans expressing Ler (pLer) or ECs1581 (pECs1581) from low copy number plasmids.

    Article Snippet: In the analysis of T3S proteins, either DMEM (Sigma-Aldrich) or MEM-HEPES (Sigma-Aldrich) supplemented with 250 nM Fe(NO3 )2 and 0.2% glucose was used for culturing strains.

    Techniques: Activity Assay, Mutagenesis, Clone Assay, Fluorescence, Plasmid Preparation, Transformation Assay, Cell Culture, Produced, Expressing, Low Copy Number, Reverse Transcription Polymerase Chain Reaction, Western Blot

    Tim4-affinity purification of sEVs. ( a ) Thioglycollate-elicited mouse peritoneal macrophages (pMac) (3 × 10 5 cells/ml) were cultured in 10 ml of DMEM containing 5% EV-depleted FBS for 48 h. 10K sup of pMac (4 ml each) were incubated with Dynabeads or mouse Tim4-Fc-conjugated Dynabeads overnight. Bound sEVs were eluted, subjected to SDS-PAGE and analyzed by western blot (right panel) with anti-CD63, anti-CD9, or anti-CD81 antibody. Total proteins in the sEV fractions were visualized by Oriole fluorescent gel stain (left panel). The amount loaded in the input lane was equivalent to 1/1000 volume of the total supernatant. ( b , c ) sEVs were purified from 10K sup (4 ml of medium containing 5% EV-depleted FBS) of K562 cells (1 × 10 6 cells/ml) ( b ) or pMac (4 × 10 5 cells/ml) ( c ) by each method. The purified sEV fractions were adjusted to the same volume (60 μl), subjected to SDS-PAGE, and analyzed by Oriole stain (left panels) or western blot (right panels). TEI: Total Exosome Isolation. The number of sEV particles in the purified fractions were also analyzed by NTA assay using NanoSight LM10 (Malvern), and the calculated purification recovery yield relative to total sEV particles was plotted in the lower graphs. ( d , e ) Protein concentration of purified sEV fractions from K562 cells ( d ) or pMac ( e ) was determined by BCA protein assay, and adjusted to the same in each method. Equal amount of proteins (100 ng) were subjected to SDS-PAGE, and analyzed by Oriole stain (left panels) or western blot (right panels). ( f ) The flow through of 10K sup of K562 cells after Tim4-affinity purification was further ultracentrifuged, and the same proportion of the protein amount to the total purified protein amount as Tim4-affinity purification was loaded to detect residual exosomes by western blot with anti-CD63 antibody (Tim4 FT → UC). ( g , h ) sEVs were isolated from 50 μl of mouse serum ( g ) or 500 μl of human urine ( h ) by each method, adjusted to the same volume and analyzed by western blot.

    Journal: Scientific Reports

    Article Title: A novel affinity-based method for the isolation of highly purified extracellular vesicles

    doi: 10.1038/srep33935

    Figure Lengend Snippet: Tim4-affinity purification of sEVs. ( a ) Thioglycollate-elicited mouse peritoneal macrophages (pMac) (3 × 10 5 cells/ml) were cultured in 10 ml of DMEM containing 5% EV-depleted FBS for 48 h. 10K sup of pMac (4 ml each) were incubated with Dynabeads or mouse Tim4-Fc-conjugated Dynabeads overnight. Bound sEVs were eluted, subjected to SDS-PAGE and analyzed by western blot (right panel) with anti-CD63, anti-CD9, or anti-CD81 antibody. Total proteins in the sEV fractions were visualized by Oriole fluorescent gel stain (left panel). The amount loaded in the input lane was equivalent to 1/1000 volume of the total supernatant. ( b , c ) sEVs were purified from 10K sup (4 ml of medium containing 5% EV-depleted FBS) of K562 cells (1 × 10 6 cells/ml) ( b ) or pMac (4 × 10 5 cells/ml) ( c ) by each method. The purified sEV fractions were adjusted to the same volume (60 μl), subjected to SDS-PAGE, and analyzed by Oriole stain (left panels) or western blot (right panels). TEI: Total Exosome Isolation. The number of sEV particles in the purified fractions were also analyzed by NTA assay using NanoSight LM10 (Malvern), and the calculated purification recovery yield relative to total sEV particles was plotted in the lower graphs. ( d , e ) Protein concentration of purified sEV fractions from K562 cells ( d ) or pMac ( e ) was determined by BCA protein assay, and adjusted to the same in each method. Equal amount of proteins (100 ng) were subjected to SDS-PAGE, and analyzed by Oriole stain (left panels) or western blot (right panels). ( f ) The flow through of 10K sup of K562 cells after Tim4-affinity purification was further ultracentrifuged, and the same proportion of the protein amount to the total purified protein amount as Tim4-affinity purification was loaded to detect residual exosomes by western blot with anti-CD63 antibody (Tim4 FT → UC). ( g , h ) sEVs were isolated from 50 μl of mouse serum ( g ) or 500 μl of human urine ( h ) by each method, adjusted to the same volume and analyzed by western blot.

    Article Snippet: The pMac were obtained 3 days after injection of 2 ml of 3% brewer thioglycollate medium (Sigma-Aldrich) into the peritoneal cavity of C57BL/6 mice (10-week-old female mice, SLC, Japan). pMac were cultured in DMEM (Nacalai Tesque, Japan) supplemented with 5% or 10% EV-depleted FBS for 48 h. The cell conditioned medium was collected and sequentially centrifuged at 300× g for 10 min, 2,000×g for 20 min, and 10,000× g for 30 min and filtered through 0.22 μm Millex-GV filter (Merck Millipore) to remove cells, cellular debris, and large EVs, resulting in the final pre-cleared cell culture supernatant (10K sup).

    Techniques: Affinity Purification, Cell Culture, Incubation, SDS Page, Western Blot, Staining, Purification, Isolation, Protein Concentration, BIA-KA, Flow Cytometry