methyl β cyclodextrin  (Millipore)


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    Name:
    Methyl beta cyclodextrin
    Description:
    Methyl β cyclodextrin is a heptasaccharide soluble in water and has more affinity to cholesterol due to the presence of hydrophobic core
    Catalog Number:
    c4555
    Price:
    None
    Applications:
    Methyl-β-cyclodextrin has been used:. to study the effect of cholesterol depletion, from sperm membrane on sperm's ability to undergo acrosome reaction.. to determine the effect of caveolin overexpression and its loss on pro-survival and pro-growth signaling. in conventional in vitro fertilization
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    Structured Review

    Millipore methyl β cyclodextrin
    Methyl beta cyclodextrin
    Methyl β cyclodextrin is a heptasaccharide soluble in water and has more affinity to cholesterol due to the presence of hydrophobic core
    https://www.bioz.com/result/methyl β cyclodextrin/product/Millipore
    Average 99 stars, based on 63 article reviews
    Price from $9.99 to $1999.99
    methyl β cyclodextrin - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "Complexation of C6-Ceramide with Cholesteryl Phosphocholine - A Potent Solvent-Free Ceramide Delivery Formulation for Cells in Culture"

    Article Title: Complexation of C6-Ceramide with Cholesteryl Phosphocholine - A Potent Solvent-Free Ceramide Delivery Formulation for Cells in Culture

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0061290

    Effect of C6-Cer on cell proliferation. A. FRTL-5 cells were preincubated for 48 h with 0.05 mM C6-Cer/CholPC or C6-Cer/DMSO. [ 3 H]Thymidine incorporation into cellular DNA during the last 4 h was determined. B. Cell proliferation measurement using cell count. Effect of C6-Cer on cell proliferation on FRTL-5 was measured by counting the cells after preincubation for 48 h with 0.05 mM C6-Cer/CholPC or C6-Cer/DMSO. DMSO alone was used as control. Each value gives the amount of cells per plate. C. HeLa cells were incubated with 0.05 mM C6-Cer/CholPC or C6-Cer/DMSO for 12 h. [ 3 H]Thymidine incorporation into cellular DNA during the last 4 h was determined. D. FRTL-5 cells were exposed to 0.05 mM L-erythro-C6-Cer/CholPC or C6-dihydroCer/CholPC for 24 h after which [ 3 H]thymidine incorporation into cellular DNA during the last 4 h was determined. E. FRTL-5 cells were exposed for 48 h to 0.05 mM Chol/CholPC, Chol/DMSO, or Chol/m-β-cyclodextrin after which [ 3 H]thymidine incorporation into cellular DNA during the last 4 h was determined. Each value is the mean ± SEM of at least 3 independent experiments. *P
    Figure Legend Snippet: Effect of C6-Cer on cell proliferation. A. FRTL-5 cells were preincubated for 48 h with 0.05 mM C6-Cer/CholPC or C6-Cer/DMSO. [ 3 H]Thymidine incorporation into cellular DNA during the last 4 h was determined. B. Cell proliferation measurement using cell count. Effect of C6-Cer on cell proliferation on FRTL-5 was measured by counting the cells after preincubation for 48 h with 0.05 mM C6-Cer/CholPC or C6-Cer/DMSO. DMSO alone was used as control. Each value gives the amount of cells per plate. C. HeLa cells were incubated with 0.05 mM C6-Cer/CholPC or C6-Cer/DMSO for 12 h. [ 3 H]Thymidine incorporation into cellular DNA during the last 4 h was determined. D. FRTL-5 cells were exposed to 0.05 mM L-erythro-C6-Cer/CholPC or C6-dihydroCer/CholPC for 24 h after which [ 3 H]thymidine incorporation into cellular DNA during the last 4 h was determined. E. FRTL-5 cells were exposed for 48 h to 0.05 mM Chol/CholPC, Chol/DMSO, or Chol/m-β-cyclodextrin after which [ 3 H]thymidine incorporation into cellular DNA during the last 4 h was determined. Each value is the mean ± SEM of at least 3 independent experiments. *P

    Techniques Used: Cell Counting, Incubation

    2) Product Images from "Vibrio vulnificus VvpE inhibits mucin 2 expression by hypermethylation via lipid raft-mediated ROS signaling in intestinal epithelial cells"

    Article Title: Vibrio vulnificus VvpE inhibits mucin 2 expression by hypermethylation via lipid raft-mediated ROS signaling in intestinal epithelial cells

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2015.152

    Role of VvpE and its related signal molecules on Muc2 production. ( a ) HT29-MTX cells were pretreated with M β CD, NAC, and 5-azacytidine prior to rVvpE (50 pg/ml) exposure for 4 h. The level of Muc2 protein was quantified by ELISA. Error bars represent the means±S.E. ( n =5). * P
    Figure Legend Snippet: Role of VvpE and its related signal molecules on Muc2 production. ( a ) HT29-MTX cells were pretreated with M β CD, NAC, and 5-azacytidine prior to rVvpE (50 pg/ml) exposure for 4 h. The level of Muc2 protein was quantified by ELISA. Error bars represent the means±S.E. ( n =5). * P

    Techniques Used: Enzyme-linked Immunosorbent Assay

    3) Product Images from "Inhibiting mevalonate pathway enzymes increases stromal cell resilience to a cholesterol-dependent cytolysin"

    Article Title: Inhibiting mevalonate pathway enzymes increases stromal cell resilience to a cholesterol-dependent cytolysin

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-17138-y

    Inhibition of mevalonate pathway enzymes. ( A , C ) BESC from 3 animals and ( B , D ) HESC from 3 independent passages were incubated in control serum-free medium, or media containing 1 μM atorvastatin (AT), 10 μM alendronate (AL), 10 μM zaragozic acid (ZA) or methyl-β-cyclodextrin (MBCD) for 48 h. ( A , B ) Cell viability was assessed by MTT assay. Data are expressed as mean (SEM), and were analyzed by one-way ANOVA and Dunnett’s multiple comparison post hoc test; values differ from control, *P
    Figure Legend Snippet: Inhibition of mevalonate pathway enzymes. ( A , C ) BESC from 3 animals and ( B , D ) HESC from 3 independent passages were incubated in control serum-free medium, or media containing 1 μM atorvastatin (AT), 10 μM alendronate (AL), 10 μM zaragozic acid (ZA) or methyl-β-cyclodextrin (MBCD) for 48 h. ( A , B ) Cell viability was assessed by MTT assay. Data are expressed as mean (SEM), and were analyzed by one-way ANOVA and Dunnett’s multiple comparison post hoc test; values differ from control, *P

    Techniques Used: Inhibition, Incubation, MTT Assay

    The mevalonate pathway leads to cholesterol synthesis. Acetoacetyl CoA and acetyl CoA are converted to squalene, which is subsequently converted to cholesterol. Important enzymes in the pathway include 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR), farnesyl pyrophosphate synthase (FDPS), and farnesyl diphosphate farnesyltransferase 1 (FDFT1, also commonly called squalene synthase). Each of these enzymes can be inhibited by statins, bisphosphonates, and zaragozic acid, respectively; cholesterol can be depleted using methyl-β-cyclodextrin (MBCD).
    Figure Legend Snippet: The mevalonate pathway leads to cholesterol synthesis. Acetoacetyl CoA and acetyl CoA are converted to squalene, which is subsequently converted to cholesterol. Important enzymes in the pathway include 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR), farnesyl pyrophosphate synthase (FDPS), and farnesyl diphosphate farnesyltransferase 1 (FDFT1, also commonly called squalene synthase). Each of these enzymes can be inhibited by statins, bisphosphonates, and zaragozic acid, respectively; cholesterol can be depleted using methyl-β-cyclodextrin (MBCD).

    Techniques Used:

    Mevalonate pathway inhibitors and cellular resilience to PLO. ( A , C , E ) BESC from 3 animals, and ( B , D , F ) HESC from 3 independent passages were incubated with control serum-free media (0), or media containing 1 μM atorvastatin (AT), 10 μM alendronate (AL), 10 μM zaragozic acid (ZA) or methyl-β-cyclodextrin (MBCD) for 48 h, and then challenged with control media (■) or media containing PLO ( ) using 100 HU/ml for BESC and 200 HU/ml for HESC, for 2 h. Cell viability was evaluated by MTT assay ( A, B ), supernatants were collected for LDH assay ( C , D ), and total cell DNA was determined by CyQuant assay ( E , F ). Data are expressed as mean (SEM), and were analyzed by one-way ANOVA with Dunnett’s multiple comparison post hoc test; values differ from PLO treatment with no inhibitor, *P
    Figure Legend Snippet: Mevalonate pathway inhibitors and cellular resilience to PLO. ( A , C , E ) BESC from 3 animals, and ( B , D , F ) HESC from 3 independent passages were incubated with control serum-free media (0), or media containing 1 μM atorvastatin (AT), 10 μM alendronate (AL), 10 μM zaragozic acid (ZA) or methyl-β-cyclodextrin (MBCD) for 48 h, and then challenged with control media (■) or media containing PLO ( ) using 100 HU/ml for BESC and 200 HU/ml for HESC, for 2 h. Cell viability was evaluated by MTT assay ( A, B ), supernatants were collected for LDH assay ( C , D ), and total cell DNA was determined by CyQuant assay ( E , F ). Data are expressed as mean (SEM), and were analyzed by one-way ANOVA with Dunnett’s multiple comparison post hoc test; values differ from PLO treatment with no inhibitor, *P

    Techniques Used: Incubation, MTT Assay, Lactate Dehydrogenase Assay, CyQUANT Assay

    4) Product Images from "High-resolution imaging and quantification of plasma membrane cholesterol by NanoSIMS"

    Article Title: High-resolution imaging and quantification of plasma membrane cholesterol by NanoSIMS

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1621432114

    NanoSIMS analysis of [ 15 N]ALO-D4 binding to the plasma membrane of CHO-KI cells grown under standard conditions. CHO-K1 cells (imaged in A – C ) were plated on silicon wafers and grown overnight and given either no treatment ( A ); treated with 10 mM methyl-β-cyclodextrin (+MβCD) for 15 min at 37 °C ( B ); or treated with 100 milliunits/mL of sphingomyelinase for 30 min at 37 °C (+SMase) ( C ). The cells were then washed and incubated with 20 μg/mL [ 15 N]ALO-D4 for 2 h at 4 °C. NanoSIMS images were generated based on 12 C 14 N – ions (to visualize cell morphology) and on the 15 N/ 14 N ratio. (Scale bar, 10 μm.) The color scale shows the range of 15 N/ 14 N ratios. ( D ) 15 N/ 14 N ratios in microvilli (black solid circles) and nonmicrovilli regions (red solid circles) ( n = 30) of the plasma membrane of two nontreated (NT) and SMase-treated (SMase) CHO-K1 cells. ( E and F ) Bar graphs depicting 15 N/ 14 N ratios in microvilli and nonmicrovilli regions of the plasma membrane in NT and SMase-treated cells. Data were analyzed with an unpaired Student’s t test with Welch’s correction.
    Figure Legend Snippet: NanoSIMS analysis of [ 15 N]ALO-D4 binding to the plasma membrane of CHO-KI cells grown under standard conditions. CHO-K1 cells (imaged in A – C ) were plated on silicon wafers and grown overnight and given either no treatment ( A ); treated with 10 mM methyl-β-cyclodextrin (+MβCD) for 15 min at 37 °C ( B ); or treated with 100 milliunits/mL of sphingomyelinase for 30 min at 37 °C (+SMase) ( C ). The cells were then washed and incubated with 20 μg/mL [ 15 N]ALO-D4 for 2 h at 4 °C. NanoSIMS images were generated based on 12 C 14 N – ions (to visualize cell morphology) and on the 15 N/ 14 N ratio. (Scale bar, 10 μm.) The color scale shows the range of 15 N/ 14 N ratios. ( D ) 15 N/ 14 N ratios in microvilli (black solid circles) and nonmicrovilli regions (red solid circles) ( n = 30) of the plasma membrane of two nontreated (NT) and SMase-treated (SMase) CHO-K1 cells. ( E and F ) Bar graphs depicting 15 N/ 14 N ratios in microvilli and nonmicrovilli regions of the plasma membrane in NT and SMase-treated cells. Data were analyzed with an unpaired Student’s t test with Welch’s correction.

    Techniques Used: Binding Assay, Incubation, Generated

    NanoSIMS analysis of [ 15 . Coverslips received no treatment ( A ); treatment with 10 mM methyl-β-cyclodextrin for 15 min at 37 °C (+MβCD) ( B ); or treatment with 100 milliunits/mL of sphingomyelinase for 30 min at 37 °C (+SMase) ( C ). The cells were then washed and incubated with 20 μg/mL [ 15 N]ALO-D4 at 4 °C for 2 h. Next, the cells were fixed, dehydrated, resin embedded, and sectioned. NanoSIMS images were generated based on 12 C 14 N − secondary ions (to define cell morphology) and 15 N/ 14 N ratios {to visualize binding of [ 15 N]ALO-D4}. Peaks in 15 N/ 14 N ratios on the line graphs are centered above the plasma membrane. (Scale bar, 10 μm.) ( D – F ) Line graphs showing 15 N/ 14 N ratios across cells.
    Figure Legend Snippet: NanoSIMS analysis of [ 15 . Coverslips received no treatment ( A ); treatment with 10 mM methyl-β-cyclodextrin for 15 min at 37 °C (+MβCD) ( B ); or treatment with 100 milliunits/mL of sphingomyelinase for 30 min at 37 °C (+SMase) ( C ). The cells were then washed and incubated with 20 μg/mL [ 15 N]ALO-D4 at 4 °C for 2 h. Next, the cells were fixed, dehydrated, resin embedded, and sectioned. NanoSIMS images were generated based on 12 C 14 N − secondary ions (to define cell morphology) and 15 N/ 14 N ratios {to visualize binding of [ 15 N]ALO-D4}. Peaks in 15 N/ 14 N ratios on the line graphs are centered above the plasma membrane. (Scale bar, 10 μm.) ( D – F ) Line graphs showing 15 N/ 14 N ratios across cells.

    Techniques Used: Incubation, Generated, Binding Assay

    NanoSIMS imaging of [ 15 ). The cells were then washed and grown without supplemental cholesterol for 44 h. Next, the cells were plated on silicon wafers, grown overnight, and then given no treatment ( A ); treated with 10 mM methyl-β-cyclodextrin for 15 min at 37 °C (+MβCD) ( B ); or treated with 100 milliunits/mL of sphingomyelinase for 30 min at 37 °C (+SMase) ( C ). The cells were then washed and incubated with 20 μg/mL [ 15 N]ALO-D4 for 2 h at 4 °C. NanoSIMS images were generated based on secondary electrons (SEs) and on the ratio of 12 C 15 N – to 12 C 14 N – secondary ions ( 15 N/ 14 N). (Scale bar, 10 μm.) The color scale shows the range of 15 N/ 14 N ratios. ( D ) 15 N/ 14 N ratios in microvilli (black solid circles) and nonmicrovilli regions (red solid circles) ( n = 30) of two nontreated (NT) and SMase-treated (SMase) cells. ( E and F ) Bar graphs of 15 N/ 14 N ratios in microvilli and nonmicrovilli regions on the plasma membrane of NT and SMase-treated cells. Data were analyzed with an unpaired Student’s t test with Welch’s correction.
    Figure Legend Snippet: NanoSIMS imaging of [ 15 ). The cells were then washed and grown without supplemental cholesterol for 44 h. Next, the cells were plated on silicon wafers, grown overnight, and then given no treatment ( A ); treated with 10 mM methyl-β-cyclodextrin for 15 min at 37 °C (+MβCD) ( B ); or treated with 100 milliunits/mL of sphingomyelinase for 30 min at 37 °C (+SMase) ( C ). The cells were then washed and incubated with 20 μg/mL [ 15 N]ALO-D4 for 2 h at 4 °C. NanoSIMS images were generated based on secondary electrons (SEs) and on the ratio of 12 C 15 N – to 12 C 14 N – secondary ions ( 15 N/ 14 N). (Scale bar, 10 μm.) The color scale shows the range of 15 N/ 14 N ratios. ( D ) 15 N/ 14 N ratios in microvilli (black solid circles) and nonmicrovilli regions (red solid circles) ( n = 30) of two nontreated (NT) and SMase-treated (SMase) cells. ( E and F ) Bar graphs of 15 N/ 14 N ratios in microvilli and nonmicrovilli regions on the plasma membrane of NT and SMase-treated cells. Data were analyzed with an unpaired Student’s t test with Welch’s correction.

    Techniques Used: Imaging, Incubation, Generated

    5) Product Images from "Plasma membrane order and fluidity are diversely triggered by elicitors of plant defence"

    Article Title: Plasma membrane order and fluidity are diversely triggered by elicitors of plant defence

    Journal: Journal of Experimental Botany

    doi: 10.1093/jxb/erw284

    Sterol trapping enhances ROS production induced by the elicitation signalling cascade. (A) Fluidity is enhanced by sterol depletion. Half-maximal time of fluorescence recovery was measured by FRAP experiments after sterol depletion (5min of a 50nM cryptogein elicitation, cry, or 15min of a 5mM methyl-β-cyclodextrin treatment, MβCD) and/or phosphorylation inhibition (by a 5min incubation with 2.5mM staurosporin, Stau). (B and C) Early events of the signalling cascade were measured in control conditions (no addition in the medium, –) and after elicitation treatment with either 50nM cryptogein (Cry) or 50ng ml −1 oligogalacturonides (OG). A pre-incubation with (grey histogram) or without (black histogram) 5mM MβCD was performed to evaluate the effect of sterol trapping on these parameters. (B) ROS production measurement. The sum of the ROS production during the first 60min was measured by chemiluminescence and reported relative to the elicitor-induced treatment value (without sterol depletion). (C) pH alkalinization was evaluated after 30min of MβCD treatment. Mean values ±SEM ( n > 5 experiments). Letters indicate a significant difference between treatments ( P -value
    Figure Legend Snippet: Sterol trapping enhances ROS production induced by the elicitation signalling cascade. (A) Fluidity is enhanced by sterol depletion. Half-maximal time of fluorescence recovery was measured by FRAP experiments after sterol depletion (5min of a 50nM cryptogein elicitation, cry, or 15min of a 5mM methyl-β-cyclodextrin treatment, MβCD) and/or phosphorylation inhibition (by a 5min incubation with 2.5mM staurosporin, Stau). (B and C) Early events of the signalling cascade were measured in control conditions (no addition in the medium, –) and after elicitation treatment with either 50nM cryptogein (Cry) or 50ng ml −1 oligogalacturonides (OG). A pre-incubation with (grey histogram) or without (black histogram) 5mM MβCD was performed to evaluate the effect of sterol trapping on these parameters. (B) ROS production measurement. The sum of the ROS production during the first 60min was measured by chemiluminescence and reported relative to the elicitor-induced treatment value (without sterol depletion). (C) pH alkalinization was evaluated after 30min of MβCD treatment. Mean values ±SEM ( n > 5 experiments). Letters indicate a significant difference between treatments ( P -value

    Techniques Used: Fluorescence, Inhibition, Incubation

    6) Product Images from "Group A Streptococcal M Protein Activates the NLRP3 Inflammasome"

    Article Title: Group A Streptococcal M Protein Activates the NLRP3 Inflammasome

    Journal: Nature microbiology

    doi: 10.1038/s41564-017-0005-6

    M1 uptake is required for inflammasome activation a, Production of IL-1β measured by ELISA from THP1-Mϕ that were untreated (Control) or pretreated with wortmannin or methyl-β-cyclodextrin for 30 min or with Pitstop-2 for 10 min prior to stimulation with M1 protein (2 μM) for 1 h. b, Production of processed IL-1β in THP1-Mϕ that were either untreated or pretreated with Pitstop-2 for 10 min and then treated with M1 (2 μM) for 1 h. c, Confocal microscopy of BMDMs untreated (-M1) or incubated with M1-mCherry protein (2 μM) for the indicated times. DAPI (blue) and M1-mCherry (red) merged images are shown in the left panels, and a merge of fluorescence and phase contrast images are shown in the right panels; scale bar = 10 μm. d, Confocal microscopy images of BMDMs incubated for 10 min with M1-mCherry protein (2 μM) and then fixed and stained with anti-EEA1 antibody. The colocalization of M1-mCherry (red) and EEA1 (green) is indicated by white arrows in the merged image. At bottom is a magnification of the dashed boxed region in the merged image; scale bar = 10μm. Panel e, shows confocal microscopy images of BMDMs treated with M1-mCherry for 30 min in the presence (+Pitstop) or in the absence (-Pitstop) of Pitstop-2. Panel f , shows quantification of M1-mCherry fluorescence (intensity of M1-mCherry fluorescence of each treatment group and shown as relative fluorescence in arbitrary units). DAPI (blue) and M1-mCherry (red) merged images are shown; scale bar = 10μm. g, LDH release from BMDMs pretreated (+ priming) or untreated (- priming) with LPS for 4 h and incubated without (-M1) or with (+M1) M1-mCherry (2 μM) in the presence (+ Pit 2) or absence (- Pit 2) of Pitstop-2 (100 % represents total cytolysis). Data in panels a, b, and g are plotted as the mean ± SEM from three independent experiments performed in triplicate. Panels c, e, and d show representative images of three and two independent experiments, respectively. Data in panel f is plotted as the mean ± SEM, representing the fluorescence of at least 100 cells from two independent experiments. Data in panels a, b, f and g were analyzed by Student's t -test. NS = not significant (P > 0.05), **P
    Figure Legend Snippet: M1 uptake is required for inflammasome activation a, Production of IL-1β measured by ELISA from THP1-Mϕ that were untreated (Control) or pretreated with wortmannin or methyl-β-cyclodextrin for 30 min or with Pitstop-2 for 10 min prior to stimulation with M1 protein (2 μM) for 1 h. b, Production of processed IL-1β in THP1-Mϕ that were either untreated or pretreated with Pitstop-2 for 10 min and then treated with M1 (2 μM) for 1 h. c, Confocal microscopy of BMDMs untreated (-M1) or incubated with M1-mCherry protein (2 μM) for the indicated times. DAPI (blue) and M1-mCherry (red) merged images are shown in the left panels, and a merge of fluorescence and phase contrast images are shown in the right panels; scale bar = 10 μm. d, Confocal microscopy images of BMDMs incubated for 10 min with M1-mCherry protein (2 μM) and then fixed and stained with anti-EEA1 antibody. The colocalization of M1-mCherry (red) and EEA1 (green) is indicated by white arrows in the merged image. At bottom is a magnification of the dashed boxed region in the merged image; scale bar = 10μm. Panel e, shows confocal microscopy images of BMDMs treated with M1-mCherry for 30 min in the presence (+Pitstop) or in the absence (-Pitstop) of Pitstop-2. Panel f , shows quantification of M1-mCherry fluorescence (intensity of M1-mCherry fluorescence of each treatment group and shown as relative fluorescence in arbitrary units). DAPI (blue) and M1-mCherry (red) merged images are shown; scale bar = 10μm. g, LDH release from BMDMs pretreated (+ priming) or untreated (- priming) with LPS for 4 h and incubated without (-M1) or with (+M1) M1-mCherry (2 μM) in the presence (+ Pit 2) or absence (- Pit 2) of Pitstop-2 (100 % represents total cytolysis). Data in panels a, b, and g are plotted as the mean ± SEM from three independent experiments performed in triplicate. Panels c, e, and d show representative images of three and two independent experiments, respectively. Data in panel f is plotted as the mean ± SEM, representing the fluorescence of at least 100 cells from two independent experiments. Data in panels a, b, f and g were analyzed by Student's t -test. NS = not significant (P > 0.05), **P

    Techniques Used: Activation Assay, Enzyme-linked Immunosorbent Assay, Confocal Microscopy, Incubation, Fluorescence, Staining

    7) Product Images from "Modulation of deregulated chaperone-mediated autophagy by a phosphopeptide"

    Article Title: Modulation of deregulated chaperone-mediated autophagy by a phosphopeptide

    Journal: Autophagy

    doi: 10.1080/15548627.2015.1017179

    B cells internalize the P140 peptide by clathrin-mediated endocytosis. B cells from MRL/lpr mice were pretreated 30 min at 37°C with the following endocytosis inhibitors: chlorpromazine (CPZ) for clathrin-mediated endocytosis ( A ), filipin for caveolin-mediated endocytosis ( B ), and methyl-β-cyclodextrin for macropinocytosis ( C ), followed by the addition of Alexa Fluor 488-labeled P140 peptide for 30 min at 37°C. TF, bodipy, and dextran were used as respective markers of each pathway. As a control, cells were incubated at 4°C to inhibit endocytosis. For macropinocytosis analysis, unpurified splenocytes were also used since dextran was not detected into B cells. Representative results of 2 different experiments are shown.
    Figure Legend Snippet: B cells internalize the P140 peptide by clathrin-mediated endocytosis. B cells from MRL/lpr mice were pretreated 30 min at 37°C with the following endocytosis inhibitors: chlorpromazine (CPZ) for clathrin-mediated endocytosis ( A ), filipin for caveolin-mediated endocytosis ( B ), and methyl-β-cyclodextrin for macropinocytosis ( C ), followed by the addition of Alexa Fluor 488-labeled P140 peptide for 30 min at 37°C. TF, bodipy, and dextran were used as respective markers of each pathway. As a control, cells were incubated at 4°C to inhibit endocytosis. For macropinocytosis analysis, unpurified splenocytes were also used since dextran was not detected into B cells. Representative results of 2 different experiments are shown.

    Techniques Used: Mouse Assay, Labeling, Incubation

    8) Product Images from "Cellular trafficking and anticancer activity of Garcinia mangostana extract-encapsulated polymeric nanoparticles"

    Article Title: Cellular trafficking and anticancer activity of Garcinia mangostana extract-encapsulated polymeric nanoparticles

    Journal: International Journal of Nanomedicine

    doi: 10.2147/IJN.S66511

    Endocytic mechanism of GME-EC/MC nanoparticles in HeLa cells. Cells were treated with various endocytic inhibitors prior to nanoparticle addition. Notes: ( A ) Chlorpromazine and sucrose 0.45 M, inhibitors of clathrin-mediated endocytosis; ( B ) genistein, an inhibitor of caveolae-mediated endocytosis; ( C ) methyl-β-cyclodextrin and lovastatin, which depleted cholesterol, and uptake of nanoparticles at 4°C; and ( D ) amiloride, an inhibitor of macropinocytosis. ** P
    Figure Legend Snippet: Endocytic mechanism of GME-EC/MC nanoparticles in HeLa cells. Cells were treated with various endocytic inhibitors prior to nanoparticle addition. Notes: ( A ) Chlorpromazine and sucrose 0.45 M, inhibitors of clathrin-mediated endocytosis; ( B ) genistein, an inhibitor of caveolae-mediated endocytosis; ( C ) methyl-β-cyclodextrin and lovastatin, which depleted cholesterol, and uptake of nanoparticles at 4°C; and ( D ) amiloride, an inhibitor of macropinocytosis. ** P

    Techniques Used:

    9) Product Images from "Corticotropin Releasing Factor-Induced CREB Activation in Striatal Neurons Occurs via a Novel G?? Signaling Pathway"

    Article Title: Corticotropin Releasing Factor-Induced CREB Activation in Striatal Neurons Occurs via a Novel G?? Signaling Pathway

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0018114

    CRFR1 couples to Gα s in striatal neurons. (A) Pre-treatment with pertussis toxin (PTX; 500 ng/mL) did not affect CRF-induced pCREB ( F = 11.10). (B) CTX blocked CRF-induced CREB phosphorylation, but had no effect on MAPK-dependent CREB phosphorylation induced by depolarization (60 K; F = 35.15). (C) CRF (4 µM) increased cAMP concentrations in the presence of the cholesterol chelator methyl-β-cyclodextrin (MβCD; 10 mM).
    Figure Legend Snippet: CRFR1 couples to Gα s in striatal neurons. (A) Pre-treatment with pertussis toxin (PTX; 500 ng/mL) did not affect CRF-induced pCREB ( F = 11.10). (B) CTX blocked CRF-induced CREB phosphorylation, but had no effect on MAPK-dependent CREB phosphorylation induced by depolarization (60 K; F = 35.15). (C) CRF (4 µM) increased cAMP concentrations in the presence of the cholesterol chelator methyl-β-cyclodextrin (MβCD; 10 mM).

    Techniques Used:

    10) Product Images from "ER Stress-Induced Clearance of Misfolded GPI-Anchored Proteins via the Secretory Pathway"

    Article Title: ER Stress-Induced Clearance of Misfolded GPI-Anchored Proteins via the Secretory Pathway

    Journal: Cell

    doi: 10.1016/j.cell.2014.06.026

    PrP ∗ Traffics to the Plasma Membrane during RESET (A) Time-lapse images of YFP-PrP ∗ -expressing cells treated with TG + methyl-β-cyclodextrin (MβCD) (top) or TG alone (bottom) taken at two different focal planes. Mid-cell indicates a focal plane at the widest point of the nucleus and coverslip indicates a focal plane close to the coverslip where the largest portion of the plasma membrane is in focus. (B) YFP-PrP ∗ cells were untreated or treated with TG and MβCD and then fixed after 90 min and stained with anti-GFP antibody without permeabilization to detect cell surface YFP-PrP ∗ . (C) Antibody uptake assay for internalization of YFP-PrP ∗ from the plasma membrane. YFP-PrP ∗ -expressing or untransfected (Untf’d) cells were treated for 1 hr with TG in the presence of leupeptin and either anti-GFP or anti-Myc. The cells were then washed, fixed, permeabilized, and stained with Cy5-conjugated secondary antibody to detect internalized antibody. Scale bars, 10 μm.
    Figure Legend Snippet: PrP ∗ Traffics to the Plasma Membrane during RESET (A) Time-lapse images of YFP-PrP ∗ -expressing cells treated with TG + methyl-β-cyclodextrin (MβCD) (top) or TG alone (bottom) taken at two different focal planes. Mid-cell indicates a focal plane at the widest point of the nucleus and coverslip indicates a focal plane close to the coverslip where the largest portion of the plasma membrane is in focus. (B) YFP-PrP ∗ cells were untreated or treated with TG and MβCD and then fixed after 90 min and stained with anti-GFP antibody without permeabilization to detect cell surface YFP-PrP ∗ . (C) Antibody uptake assay for internalization of YFP-PrP ∗ from the plasma membrane. YFP-PrP ∗ -expressing or untransfected (Untf’d) cells were treated for 1 hr with TG in the presence of leupeptin and either anti-GFP or anti-Myc. The cells were then washed, fixed, permeabilized, and stained with Cy5-conjugated secondary antibody to detect internalized antibody. Scale bars, 10 μm.

    Techniques Used: Expressing, Staining

    11) Product Images from "Isoprenoids increase bovine endometrial stromal cell tolerance to the cholesterol-dependent cytolysin from Trueperella pyogenes †"

    Article Title: Isoprenoids increase bovine endometrial stromal cell tolerance to the cholesterol-dependent cytolysin from Trueperella pyogenes †

    Journal: Biology of Reproduction

    doi: 10.1093/biolre/ioy099

    Cellular sensitivity to pyolysin. (A) Bovine endometrial stromal cells were incubated with culture medium containing vehicle, or molecules that target MAPK (5 μM ERK inhibitor, 10 μM JNK II inhibitor, 10 μM p38 inhibitor), cell cycle (50 μM PNU112455A, 80 μM roscovitine, 3 μM butyrolactone I), metabolic signaling pathways (10 μM AICAR, 100 μM compound C, 2 μM Torin 1, 4 μM rapamycin, 40 μM AKT inhibitor IV, 2.8 μM LY294002), or the cholesterol synthesis pathway (1 μM atorvastatin, 100 μM etidronate, 10 μM alendronate, 10 μM zaragozic acid), with 0.5 mM methyl-β-cyclodextrin as a positive control. Cells were then challenged with control medium, black bar ( ) or medium containing 100 HU/ml pyolysin red bars ( ), and cell viability was determined by MTT assay. Data are from at least three animals per treatment, and expressed as percent cell viability compared with cells in control medium. Data are presented as mean (SEM), and were analyzed by one-way ANOVA and Dunnett multiple comparison post hoc test; values differ from vehicle, * P
    Figure Legend Snippet: Cellular sensitivity to pyolysin. (A) Bovine endometrial stromal cells were incubated with culture medium containing vehicle, or molecules that target MAPK (5 μM ERK inhibitor, 10 μM JNK II inhibitor, 10 μM p38 inhibitor), cell cycle (50 μM PNU112455A, 80 μM roscovitine, 3 μM butyrolactone I), metabolic signaling pathways (10 μM AICAR, 100 μM compound C, 2 μM Torin 1, 4 μM rapamycin, 40 μM AKT inhibitor IV, 2.8 μM LY294002), or the cholesterol synthesis pathway (1 μM atorvastatin, 100 μM etidronate, 10 μM alendronate, 10 μM zaragozic acid), with 0.5 mM methyl-β-cyclodextrin as a positive control. Cells were then challenged with control medium, black bar ( ) or medium containing 100 HU/ml pyolysin red bars ( ), and cell viability was determined by MTT assay. Data are from at least three animals per treatment, and expressed as percent cell viability compared with cells in control medium. Data are presented as mean (SEM), and were analyzed by one-way ANOVA and Dunnett multiple comparison post hoc test; values differ from vehicle, * P

    Techniques Used: Incubation, Positive Control, MTT Assay

    (A) Endometrial stromal cells were incubated in control serum-free medium, or medium containing 20 μM farnesyl pyrophosphate, 20 μM geranylgeranyl pyrophosphate, or 0.5 mM methyl-β-cyclodextrin, and then challenged with control serum-free medium black bars ( ) or medium containing 100 HU/ml pyolysin red bars ( ) for 2 h. Cellular leakage was assessed by measuring LDH in cell supernatants, and data expressed as percent of control challenged with pyolysin. Data are presented as mean (SEM); n = 3 animals; ND, not detectable. Data were analyzed by one-way ANOVA and Dunnett multiple comparison post hoc test; values differ from pyolysin treatment, * P
    Figure Legend Snippet: (A) Endometrial stromal cells were incubated in control serum-free medium, or medium containing 20 μM farnesyl pyrophosphate, 20 μM geranylgeranyl pyrophosphate, or 0.5 mM methyl-β-cyclodextrin, and then challenged with control serum-free medium black bars ( ) or medium containing 100 HU/ml pyolysin red bars ( ) for 2 h. Cellular leakage was assessed by measuring LDH in cell supernatants, and data expressed as percent of control challenged with pyolysin. Data are presented as mean (SEM); n = 3 animals; ND, not detectable. Data were analyzed by one-way ANOVA and Dunnett multiple comparison post hoc test; values differ from pyolysin treatment, * P

    Techniques Used: Incubation

    Cellular cholesterol. (A) Endometrial stromal cells were incubated in control serum-free medium, or medium containing 20 μM farnesyl pyrophosphate, 20 μM geranylgeranyl pyrophosphate, or 0.5 mM methyl-β-cyclodextrin for 48 h. (B) Cells were incubated for 48 h in medium containing scramble siRNA or siRNA targeting FDPS or GGPS1 , or cultured with 0.5 mM methyl-β-cyclodextrin as a positive control. Cellular cholesterol was measured and normalized to phospholipid concentrations. Data are presented as mean (SEM); n = 4 independent animals for each experiment. Data were analyzed by one-way ANOVA and Dunnett multiple comparison post hoc test; values differ from control (A) or scramble (B), * P
    Figure Legend Snippet: Cellular cholesterol. (A) Endometrial stromal cells were incubated in control serum-free medium, or medium containing 20 μM farnesyl pyrophosphate, 20 μM geranylgeranyl pyrophosphate, or 0.5 mM methyl-β-cyclodextrin for 48 h. (B) Cells were incubated for 48 h in medium containing scramble siRNA or siRNA targeting FDPS or GGPS1 , or cultured with 0.5 mM methyl-β-cyclodextrin as a positive control. Cellular cholesterol was measured and normalized to phospholipid concentrations. Data are presented as mean (SEM); n = 4 independent animals for each experiment. Data were analyzed by one-way ANOVA and Dunnett multiple comparison post hoc test; values differ from control (A) or scramble (B), * P

    Techniques Used: Incubation, Cell Culture, Positive Control

    12) Product Images from "Differential sensitivity of types 1 and 2 cholecystokinin receptors to membrane cholesterol"

    Article Title: Differential sensitivity of types 1 and 2 cholecystokinin receptors to membrane cholesterol

    Journal: Journal of Lipid Research

    doi: 10.1194/jlr.M020065

    CCK radioligand binding to chimeric CCK2R/CCK1R receptors on cholesterol-modified cells. Shown are CCK competition binding curves for chimeric receptor constructs after cholesterol depletion with M β CD (left panels) or cholesterol enrichment
    Figure Legend Snippet: CCK radioligand binding to chimeric CCK2R/CCK1R receptors on cholesterol-modified cells. Shown are CCK competition binding curves for chimeric receptor constructs after cholesterol depletion with M β CD (left panels) or cholesterol enrichment

    Techniques Used: Binding Assay, Modification, Construct

    13) Product Images from "S1P1 Receptor Localization Confers Selectivity for Gi-mediated cAMP and Contractile Responses *-mediated cAMP and Contractile Responses * S⃞"

    Article Title: S1P1 Receptor Localization Confers Selectivity for Gi-mediated cAMP and Contractile Responses *-mediated cAMP and Contractile Responses * S⃞

    Journal:

    doi: 10.1074/jbc.M707422200

    Methyl-β-cyclodextrin treatment does not block S1P-mediated activation of ERK or Akt. WT cardiomyocytes were treated with methyl-β-cyclodextrin (1 m m ) for 1 h prior to stimulation with S1P (5 μ m ) for 5 min and then assayed for
    Figure Legend Snippet: Methyl-β-cyclodextrin treatment does not block S1P-mediated activation of ERK or Akt. WT cardiomyocytes were treated with methyl-β-cyclodextrin (1 m m ) for 1 h prior to stimulation with S1P (5 μ m ) for 5 min and then assayed for

    Techniques Used: Blocking Assay, Activation Assay

    Methyl-β-cyclodextrin treatment blocks S1P- and SEW2871-mediated inhibition of isoproterenol-stimulated cAMP accumulation. A , WT cardiomyocytes were treated with methyl-β-cyclodextrin (1 m m ) for 1 h followed by 500 μ m isobutylmethylxanthine
    Figure Legend Snippet: Methyl-β-cyclodextrin treatment blocks S1P- and SEW2871-mediated inhibition of isoproterenol-stimulated cAMP accumulation. A , WT cardiomyocytes were treated with methyl-β-cyclodextrin (1 m m ) for 1 h followed by 500 μ m isobutylmethylxanthine

    Techniques Used: Inhibition

    14) Product Images from "Role for Human Immunodeficiency Virus Type 1 Membrane Cholesterol in Viral Internalization"

    Article Title: Role for Human Immunodeficiency Virus Type 1 Membrane Cholesterol in Viral Internalization

    Journal: Journal of Virology

    doi: 10.1128/JVI.76.20.10356-10364.2002

    Assessment of viral endocytosis. GFP-labeled wild-type R9ΔE/VSV G (A), methyl-β-cyclodextrin-treated R9ΔE/VSV G (B), wild-type HIV-1 (C), and methyl-β-cyclodextrin-treated HIV-1 (D) viruses were used to infect CD4 + HeLa cells. Inocula contained equivalent amounts of reverse transcriptase corresponding to a multiplicity of infection of 10 for the wild-type untreated control. Virus internalization was performed in the presence of Texas Red-conjugated transferrin to label endosomes and examined after 20 min (A and B) or 2 h (C and D) at 37°C.
    Figure Legend Snippet: Assessment of viral endocytosis. GFP-labeled wild-type R9ΔE/VSV G (A), methyl-β-cyclodextrin-treated R9ΔE/VSV G (B), wild-type HIV-1 (C), and methyl-β-cyclodextrin-treated HIV-1 (D) viruses were used to infect CD4 + HeLa cells. Inocula contained equivalent amounts of reverse transcriptase corresponding to a multiplicity of infection of 10 for the wild-type untreated control. Virus internalization was performed in the presence of Texas Red-conjugated transferrin to label endosomes and examined after 20 min (A and B) or 2 h (C and D) at 37°C.

    Techniques Used: Labeling, Infection

    Confocal microscopic analysis of intracellular virus. GFP-labeled wild-type (A) and methyl-β-cyclodextrin-treated (B) HIV-1 particles and R9ΔE/VSV G (C) and methyl-β-cyclodextrin-treated R9ΔE/VSV G (D) viruses were used to infect CD4 + HeLa cells. Inocula contained equivalent amounts of reverse transcriptase corresponding to a multiplicity of infection of 1 for the wild-type untreated control. Virus internalization was examined after 2 h at 37°C. TAMRA-ConA was used to stain cell membranes.
    Figure Legend Snippet: Confocal microscopic analysis of intracellular virus. GFP-labeled wild-type (A) and methyl-β-cyclodextrin-treated (B) HIV-1 particles and R9ΔE/VSV G (C) and methyl-β-cyclodextrin-treated R9ΔE/VSV G (D) viruses were used to infect CD4 + HeLa cells. Inocula contained equivalent amounts of reverse transcriptase corresponding to a multiplicity of infection of 1 for the wild-type untreated control. Virus internalization was examined after 2 h at 37°C. TAMRA-ConA was used to stain cell membranes.

    Techniques Used: Labeling, Infection, Staining

    Cholesterol-depleted purified particles exhibit normal protein profile. (A) Reverse transcriptase (RT) activity in sucrose density gradient fractions of native and methyl-β-cyclodextrin-treated R9 HIV-1 virions. (B) Immunoblot analysis with the indicated antisera of pooled fractions corresponding to intact virions of untreated (0 mM methyl-β-cyclodextrin [CD]) and methyl-β-cyclodextrin-treated (10 mM methyl-β-cyclodextrin) R9. Cyp A, cyclophilin A. (C) GPI-anchored protein profile of untreated, methyl-β-cyclodextrin-treated, and phospholipase C (PLipase)-treated virions with the proaerolysin/anti-proaerolysin overlay procedure (top). The same membrane was probed by Western blotting with an anti-CA antibody (bottom).
    Figure Legend Snippet: Cholesterol-depleted purified particles exhibit normal protein profile. (A) Reverse transcriptase (RT) activity in sucrose density gradient fractions of native and methyl-β-cyclodextrin-treated R9 HIV-1 virions. (B) Immunoblot analysis with the indicated antisera of pooled fractions corresponding to intact virions of untreated (0 mM methyl-β-cyclodextrin [CD]) and methyl-β-cyclodextrin-treated (10 mM methyl-β-cyclodextrin) R9. Cyp A, cyclophilin A. (C) GPI-anchored protein profile of untreated, methyl-β-cyclodextrin-treated, and phospholipase C (PLipase)-treated virions with the proaerolysin/anti-proaerolysin overlay procedure (top). The same membrane was probed by Western blotting with an anti-CA antibody (bottom).

    Techniques Used: Purification, Activity Assay, Western Blot

    Alteration of viral membrane cholesterol strongly decreases HIV-1 infectivity. (A and B) [ 3 H]cholesterol-labeled wild-type (R9) and VSV G-pseudotyped (R9ΔE/VSV G) virions were treated with increasing concentrations of methyl-β-cyclodextrin (CD). (A) Virions were subsequently concentrated by ultracentrifugation, and the amounts of pelletable and free [ 3 H]cholesterol were determined by scintillation counting. For “pellets,” the numbers on the ordinate correspond to the ratio of pelleted to pelleted plus free cholesterol. (B) Infectivity of the above virions was determined in a single-round assay on CD4 + HeLa P4 cells. Results are representative of at least five experiments. Infectivity of the untreated virus was given in each case the arbitrary value of 100%. (C) Infectivity of wild-type (R9) and VSV G-pseudotyped (R9ΔE/VSV G) virions produced by transfection with decreasing amounts of VSV G-expressing plasmid: 10 μg and 0.5 μg for R9ΔE/VSV G0.1 and R9ΔE/VSV G.2, respectively. Viral supernatants were treated or not with 10 mM methyl-β-cyclodextrin and concentrated by ultracentrifugation, and virion infectivity was determined on HeLa P4 cells. (D) Infectivity of control and cholesterol-depleted virions purified on a sucrose gradient after treatment with 10 mM methyl-β-cyclodextrin, expressed in infectious units per unit of reverse transcriptase activity. Infectivity of R9 and R9ΔE/VSV G virions was measured on HeLa P4 cells, while R8BaL virions were tested on HeLa P4-CCR5 cells. (E) Virus supernatants were treated with increasing concentrations of nystatin and purified by ultracentrifugation through a 20% sucrose cushion. Infectivity was determined as described for panel B.
    Figure Legend Snippet: Alteration of viral membrane cholesterol strongly decreases HIV-1 infectivity. (A and B) [ 3 H]cholesterol-labeled wild-type (R9) and VSV G-pseudotyped (R9ΔE/VSV G) virions were treated with increasing concentrations of methyl-β-cyclodextrin (CD). (A) Virions were subsequently concentrated by ultracentrifugation, and the amounts of pelletable and free [ 3 H]cholesterol were determined by scintillation counting. For “pellets,” the numbers on the ordinate correspond to the ratio of pelleted to pelleted plus free cholesterol. (B) Infectivity of the above virions was determined in a single-round assay on CD4 + HeLa P4 cells. Results are representative of at least five experiments. Infectivity of the untreated virus was given in each case the arbitrary value of 100%. (C) Infectivity of wild-type (R9) and VSV G-pseudotyped (R9ΔE/VSV G) virions produced by transfection with decreasing amounts of VSV G-expressing plasmid: 10 μg and 0.5 μg for R9ΔE/VSV G0.1 and R9ΔE/VSV G.2, respectively. Viral supernatants were treated or not with 10 mM methyl-β-cyclodextrin and concentrated by ultracentrifugation, and virion infectivity was determined on HeLa P4 cells. (D) Infectivity of control and cholesterol-depleted virions purified on a sucrose gradient after treatment with 10 mM methyl-β-cyclodextrin, expressed in infectious units per unit of reverse transcriptase activity. Infectivity of R9 and R9ΔE/VSV G virions was measured on HeLa P4 cells, while R8BaL virions were tested on HeLa P4-CCR5 cells. (E) Virus supernatants were treated with increasing concentrations of nystatin and purified by ultracentrifugation through a 20% sucrose cushion. Infectivity was determined as described for panel B.

    Techniques Used: Infection, Labeling, Produced, Transfection, Expressing, Plasmid Preparation, Purification, Activity Assay

    Confocal microscopic analysis of virus attachment and internalization. GFP-labeled wild-type (A and B), methyl-β-cyclodextrin-treated (C and D), and envelope-defective (E and F) viruses were used to infect CD4 + HeLa cells. Inocula contained equivalent amounts of reverse transcriptase corresponding to a multiplicity of infection of 1 for the wild-type untreated control. (A, C, and E) Virus attachment, assessed after incubating the virus with the cells at 4°C for 1 h. (B, D, and F) Virus internalization, examined after 2 h at 37°C. CD4 staining (red) was performed after fixation and without permeabilization to label only molecules present at the cell surface.
    Figure Legend Snippet: Confocal microscopic analysis of virus attachment and internalization. GFP-labeled wild-type (A and B), methyl-β-cyclodextrin-treated (C and D), and envelope-defective (E and F) viruses were used to infect CD4 + HeLa cells. Inocula contained equivalent amounts of reverse transcriptase corresponding to a multiplicity of infection of 1 for the wild-type untreated control. (A, C, and E) Virus attachment, assessed after incubating the virus with the cells at 4°C for 1 h. (B, D, and F) Virus internalization, examined after 2 h at 37°C. CD4 staining (red) was performed after fixation and without permeabilization to label only molecules present at the cell surface.

    Techniques Used: Labeling, Infection, Staining

    15) Product Images from "Depolymerization of cortical actin inhibits UT-A1 urea transporter endocytosis but promotes forskolin-stimulated membrane trafficking"

    Article Title: Depolymerization of cortical actin inhibits UT-A1 urea transporter endocytosis but promotes forskolin-stimulated membrane trafficking

    Journal: American Journal of Physiology - Cell Physiology

    doi: 10.1152/ajpcell.00440.2011

    Actin depolymerization abrogates UT-A1 endocytosis. A : cell biotinylation experiment. UT-A1-MDCK cells were treated with 5 mM methyl-β-cyclodextrin (MCD) or 50 μg/ml concanavalin A (Con A) together with or without 1 μM latrunculin
    Figure Legend Snippet: Actin depolymerization abrogates UT-A1 endocytosis. A : cell biotinylation experiment. UT-A1-MDCK cells were treated with 5 mM methyl-β-cyclodextrin (MCD) or 50 μg/ml concanavalin A (Con A) together with or without 1 μM latrunculin

    Techniques Used:

    16) Product Images from "Tracking the virus-like particles of Macrobrachium rosenbergii nodavirus in insect cells"

    Article Title: Tracking the virus-like particles of Macrobrachium rosenbergii nodavirus in insect cells

    Journal: PeerJ

    doi: 10.7717/peerj.2947

    Effect of endosomal inhibitors on the entry of  Mr Nvc VLPs into Sf9 cells. Sf9 cells were pre-incubated with different endosomal inhibitors: (A(iii)) cytochalasin D (2 µM), (A(iv)) NH 4 Cl (10 mM), (A(v)) CPZ (50 µM), (A(vi)) methyl- β -cyclodextrin (2 mM) and (A(vii)) genistein (100 µM).  Mr Nvc VLPs labelled with NHS-fluorescein (F- Mr Nvc VLPs; 25 mg/ml) were added to each pre-treated sample and incubated for 16 h in the presence of endosomal inhibitors. Bars, 20 µm. (A(ii)) Sf9 cells added with F- Mr Nvc VLPs but without any inhibitor serve as positive control, whereas (A(i)) Sf9 cells without any inhibitor nor F- Mr Nvc VLPs serve as negative control. (B) MTT assay showing the viability of Sf9 cells in the presence of inhibitors.
    Figure Legend Snippet: Effect of endosomal inhibitors on the entry of Mr Nvc VLPs into Sf9 cells. Sf9 cells were pre-incubated with different endosomal inhibitors: (A(iii)) cytochalasin D (2 µM), (A(iv)) NH 4 Cl (10 mM), (A(v)) CPZ (50 µM), (A(vi)) methyl- β -cyclodextrin (2 mM) and (A(vii)) genistein (100 µM). Mr Nvc VLPs labelled with NHS-fluorescein (F- Mr Nvc VLPs; 25 mg/ml) were added to each pre-treated sample and incubated for 16 h in the presence of endosomal inhibitors. Bars, 20 µm. (A(ii)) Sf9 cells added with F- Mr Nvc VLPs but without any inhibitor serve as positive control, whereas (A(i)) Sf9 cells without any inhibitor nor F- Mr Nvc VLPs serve as negative control. (B) MTT assay showing the viability of Sf9 cells in the presence of inhibitors.

    Techniques Used: Incubation, Positive Control, Negative Control, MTT Assay

    17) Product Images from "Single-Quantum-Dot Tracking Reveals Altered Membrane Dynamics of an Attention-Deficit/Hyperactivity-Disorder-Derived Dopamine Transporter Coding Variant"

    Article Title: Single-Quantum-Dot Tracking Reveals Altered Membrane Dynamics of an Attention-Deficit/Hyperactivity-Disorder-Derived Dopamine Transporter Coding Variant

    Journal: ACS chemical neuroscience

    doi: 10.1021/cn500202c

    DAT 615R and DAT 615C membrane diffusion after M β CD-mediated cholesterol depletion and amphetamine treatment in Flp-In 293 cells. (A) Example trajectories. Scale: 1 pixel = 200 nm. (B and D) Comparison of diffusion coefficient distributions for single DAT 615R-QDs and DAT 615C-QDs under basal, methyl- β -cyclodextrin-treated, amphetamine-treated conditions (one-way ANOVA with Bonferroni-Dunn’s post hoc test on nontransformed data: DAT 615R basal vs DAT 615C basal, *** P
    Figure Legend Snippet: DAT 615R and DAT 615C membrane diffusion after M β CD-mediated cholesterol depletion and amphetamine treatment in Flp-In 293 cells. (A) Example trajectories. Scale: 1 pixel = 200 nm. (B and D) Comparison of diffusion coefficient distributions for single DAT 615R-QDs and DAT 615C-QDs under basal, methyl- β -cyclodextrin-treated, amphetamine-treated conditions (one-way ANOVA with Bonferroni-Dunn’s post hoc test on nontransformed data: DAT 615R basal vs DAT 615C basal, *** P

    Techniques Used: Diffusion-based Assay

    18) Product Images from "Tracking Cholesterol/Sphingomyelin-Rich Membrane Domains with the Ostreolysin A-mCherry Protein"

    Article Title: Tracking Cholesterol/Sphingomyelin-Rich Membrane Domains with the Ostreolysin A-mCherry Protein

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0092783

    Binding of mCherry-OlyA, and effects of methyl-β-cyclodextrin and sphingomyelinase on binding of OlyA-mCherry to living MDCK cells. Representative fluorescent image of living MDCK cells grown at 37°C, showing no binding of 1 μM mCherry-OlyA after a 10-min incubation (A). No binding of 1 μM OlyA-mCherry was observed with cells pre-treated for 60 min with 5 mM methyl-β-cyclodextrin (B), or for 30 min with 0.5 U/mL sphingomyelinase (C). Scale bars: 20 μm. Cells were stained as described in Materials and methods.
    Figure Legend Snippet: Binding of mCherry-OlyA, and effects of methyl-β-cyclodextrin and sphingomyelinase on binding of OlyA-mCherry to living MDCK cells. Representative fluorescent image of living MDCK cells grown at 37°C, showing no binding of 1 μM mCherry-OlyA after a 10-min incubation (A). No binding of 1 μM OlyA-mCherry was observed with cells pre-treated for 60 min with 5 mM methyl-β-cyclodextrin (B), or for 30 min with 0.5 U/mL sphingomyelinase (C). Scale bars: 20 μm. Cells were stained as described in Materials and methods.

    Techniques Used: Binding Assay, Incubation, Staining

    19) Product Images from "Cholesterol Is Required for Surface Transport of Influenza Virus Hemagglutinin "

    Article Title: Cholesterol Is Required for Surface Transport of Influenza Virus Hemagglutinin

    Journal: The Journal of Cell Biology

    doi:

    Removal of cholesterol from MDCK cells affects TGN–apical surface transport of influenza virus HA, but not basolateral VSV G transport. ( A and B ) Representative example of a TGN-to-surface transport. Filter-grown MDCK cells were treated with lovastatin/mevalonate and methyl-β-cyclodextrin as detailed in Materials and Methods. After pulse labeling with [ 35 S]methionine and a TGN block at 19.5°C, viral proteins ( A , HA ; B , VSV G ) were chased to the cell surface for different times at 37°C. Arrival of HA on the apical surface was detected by trypsin treatment. Basolateral arrival of VSV G was detected by surface immunoprecipitation. ▪, untreated control cells; □, cells treated with lovastatin/mevalonate and methyl-β-cyclodextrin. ( C and D ) Averaged data of five independent experiments (see Fig. 4 ).
    Figure Legend Snippet: Removal of cholesterol from MDCK cells affects TGN–apical surface transport of influenza virus HA, but not basolateral VSV G transport. ( A and B ) Representative example of a TGN-to-surface transport. Filter-grown MDCK cells were treated with lovastatin/mevalonate and methyl-β-cyclodextrin as detailed in Materials and Methods. After pulse labeling with [ 35 S]methionine and a TGN block at 19.5°C, viral proteins ( A , HA ; B , VSV G ) were chased to the cell surface for different times at 37°C. Arrival of HA on the apical surface was detected by trypsin treatment. Basolateral arrival of VSV G was detected by surface immunoprecipitation. ▪, untreated control cells; □, cells treated with lovastatin/mevalonate and methyl-β-cyclodextrin. ( C and D ) Averaged data of five independent experiments (see Fig. 4 ).

    Techniques Used: Labeling, Blocking Assay, Immunoprecipitation

    Removal of cholesterol from MDCK cells leads to missorting of gp-80. Filter-grown MDCK cells were treated with lovastatin/mevalonate and methyl-β-cyclodextrin as detailed in Materials and Methods. After pulse labeling for 15 min with [ 35 S]methionine and a TGN block at 19.5°C, gp-80 was allowed to be secreted for 20 and 40 min at 37°C, and was recovered from apical and basolateral media, as well as from the cells by immunoprecipitation. The immunoprecipitates were analyzed by nonreducing SDS-PAGE, followed by quantitation with a PhosphorImager. ( A ) Untreated control cells. ( B ) Cells treated with lovastatin/mevalonate and methyl-β-cyclodextrin. ▪, gp-80 secreted apically; □, gp-80, secreted basolaterally.
    Figure Legend Snippet: Removal of cholesterol from MDCK cells leads to missorting of gp-80. Filter-grown MDCK cells were treated with lovastatin/mevalonate and methyl-β-cyclodextrin as detailed in Materials and Methods. After pulse labeling for 15 min with [ 35 S]methionine and a TGN block at 19.5°C, gp-80 was allowed to be secreted for 20 and 40 min at 37°C, and was recovered from apical and basolateral media, as well as from the cells by immunoprecipitation. The immunoprecipitates were analyzed by nonreducing SDS-PAGE, followed by quantitation with a PhosphorImager. ( A ) Untreated control cells. ( B ) Cells treated with lovastatin/mevalonate and methyl-β-cyclodextrin. ▪, gp-80 secreted apically; □, gp-80, secreted basolaterally.

    Techniques Used: Labeling, Blocking Assay, Immunoprecipitation, SDS Page, Quantitation Assay

    Treatment of BHK and MDCK cells with methyl- β-cyclodextrin leads to increased Triton X-100 solubility of influenza virus HA. BHK cells ( A ) and filter-grown MDCK cells ( B ) were treated with lovastatin/mevalonate and methyl-β-cyclodextrin as detailed in Materials and Methods. The cells then were extracted on ice with 1% Triton X-100, followed by centrifugation at 120,000 g to obtain detergent-soluble and -insoluble fractions. ▪, untreated control cells; □, cells treated with lovastatin/mevalonate and methyl-β-cyclodextrin.
    Figure Legend Snippet: Treatment of BHK and MDCK cells with methyl- β-cyclodextrin leads to increased Triton X-100 solubility of influenza virus HA. BHK cells ( A ) and filter-grown MDCK cells ( B ) were treated with lovastatin/mevalonate and methyl-β-cyclodextrin as detailed in Materials and Methods. The cells then were extracted on ice with 1% Triton X-100, followed by centrifugation at 120,000 g to obtain detergent-soluble and -insoluble fractions. ▪, untreated control cells; □, cells treated with lovastatin/mevalonate and methyl-β-cyclodextrin.

    Techniques Used: Solubility, Centrifugation

    Treatment of BHK cells and of filter-grown MDCK cells with methyl-β-cyclodextrin. BHK cells were left untreated ( A ), or were grown in the presence of lovastatin/mevalonate and then treated for 30 min at 37°C with 10 mM methyl-β-cyclodextrin ( B ) as detailed in Materials and Methods, and then were stained with filipin. Digital images were acquired using 5 ( A ) and 20 ( B ) integration frames, respectively. Filter-grown MDCK cells were left untreated ( C ), or were grown in the presence of lovastatin/mevalonate and then treated for 60 min at 37°C with 10 mM methyl-β-cyclodextrin ( D ) as detailed in Materials and Methods. X–Z confocal views of cells labeled with a monoclonal antibody directed against a basolateral 58-kD protein. Bars, 10 μm.
    Figure Legend Snippet: Treatment of BHK cells and of filter-grown MDCK cells with methyl-β-cyclodextrin. BHK cells were left untreated ( A ), or were grown in the presence of lovastatin/mevalonate and then treated for 30 min at 37°C with 10 mM methyl-β-cyclodextrin ( B ) as detailed in Materials and Methods, and then were stained with filipin. Digital images were acquired using 5 ( A ) and 20 ( B ) integration frames, respectively. Filter-grown MDCK cells were left untreated ( C ), or were grown in the presence of lovastatin/mevalonate and then treated for 60 min at 37°C with 10 mM methyl-β-cyclodextrin ( D ) as detailed in Materials and Methods. X–Z confocal views of cells labeled with a monoclonal antibody directed against a basolateral 58-kD protein. Bars, 10 μm.

    Techniques Used: Staining, Labeling

    Removal of cholesterol does not affect ER-to-Golgi transport. BHK cells ( A and B ) and filter-grown MDCK cells ( C and D ) were treated with lovastatin/mevalonate and methyl-β-cyclodextrin as detailed in Materials and Methods. After pulse labeling with [ 35 S]methionine, viral proteins ( A and C , HA ; B and D , VSV G ) were chased for different times at 37°C. Arrival in the Golgi complex was monitored by acquirement of resistance to endoglycosidase H digestion. ▪, untreated control cells; □, cells treated with lovastatin/mevalonate and methyl-β-cyclodextrin.
    Figure Legend Snippet: Removal of cholesterol does not affect ER-to-Golgi transport. BHK cells ( A and B ) and filter-grown MDCK cells ( C and D ) were treated with lovastatin/mevalonate and methyl-β-cyclodextrin as detailed in Materials and Methods. After pulse labeling with [ 35 S]methionine, viral proteins ( A and C , HA ; B and D , VSV G ) were chased for different times at 37°C. Arrival in the Golgi complex was monitored by acquirement of resistance to endoglycosidase H digestion. ▪, untreated control cells; □, cells treated with lovastatin/mevalonate and methyl-β-cyclodextrin.

    Techniques Used: Labeling

    Removal of cholesterol from BHK cells affects TGN-to-surface transport of influenza virus HA, but not of VSV G. ( A and B ) Representative example of a TGN-to-surface transport. BHK cells were treated with lovastatin/mevalonate and methyl-β-cyclodextrin as detailed in Materials and Methods. After pulse labeling with [ 35 S]methionine and a TGN block at 19.5°C, viral proteins ( A , HA ; B , VSV G ) were chased to the cell surface for different times at 37°C. Arrival on the surface was detected by trypsin treatment ( HA ) or by surface immunoprecipitation ( VSV G ), respectively. ▪, untreated control cells; □, cells treated with lovastatin/mevalonate and methyl-β-cyclodextrin. ( C and D ) Averaged data of four independent experiments. Relative transport indices for HA ( C ) and VSV G ( D ) after 15 and 30 min of incubation at 37°C were calculated from four experiments. Transport in cells treated with lovastatin/mevalonate and methyl-β-cyclodextrin was expressed relative to the transport in untreated control cells at the respective time.
    Figure Legend Snippet: Removal of cholesterol from BHK cells affects TGN-to-surface transport of influenza virus HA, but not of VSV G. ( A and B ) Representative example of a TGN-to-surface transport. BHK cells were treated with lovastatin/mevalonate and methyl-β-cyclodextrin as detailed in Materials and Methods. After pulse labeling with [ 35 S]methionine and a TGN block at 19.5°C, viral proteins ( A , HA ; B , VSV G ) were chased to the cell surface for different times at 37°C. Arrival on the surface was detected by trypsin treatment ( HA ) or by surface immunoprecipitation ( VSV G ), respectively. ▪, untreated control cells; □, cells treated with lovastatin/mevalonate and methyl-β-cyclodextrin. ( C and D ) Averaged data of four independent experiments. Relative transport indices for HA ( C ) and VSV G ( D ) after 15 and 30 min of incubation at 37°C were calculated from four experiments. Transport in cells treated with lovastatin/mevalonate and methyl-β-cyclodextrin was expressed relative to the transport in untreated control cells at the respective time.

    Techniques Used: Labeling, Blocking Assay, Immunoprecipitation, Incubation

    Removal of cholesterol from MDCK cells leads to partial missorting of influenza virus HA. Filter-grown MDCK cells were treated with lovastatin/mevalonate and methyl-β-cyclodextrin as detailed in Materials and Methods. After pulse labeling with [ 35 S]methionine and a TGN block at 19.5°C, HA was chased to the cell surface for different times at 37°C. Arrival on the apical or basolateral membrane, respectively, was detected by trypsin treatment. ( A ) untreated control cells; ( B ) cells treated with lovastatin/mevalonate and methyl-β-cyclodextrin. HA1 and HA2 are the products generated from HA by trypsin cleavage. The duration of transport (in minutes) is indicated above the lanes.
    Figure Legend Snippet: Removal of cholesterol from MDCK cells leads to partial missorting of influenza virus HA. Filter-grown MDCK cells were treated with lovastatin/mevalonate and methyl-β-cyclodextrin as detailed in Materials and Methods. After pulse labeling with [ 35 S]methionine and a TGN block at 19.5°C, HA was chased to the cell surface for different times at 37°C. Arrival on the apical or basolateral membrane, respectively, was detected by trypsin treatment. ( A ) untreated control cells; ( B ) cells treated with lovastatin/mevalonate and methyl-β-cyclodextrin. HA1 and HA2 are the products generated from HA by trypsin cleavage. The duration of transport (in minutes) is indicated above the lanes.

    Techniques Used: Labeling, Blocking Assay, Generated

    Treatment of BHK and MDCK cells with methyl- β-cyclodextrin leads to efficient removal of cellular cholesterol. BHK cells ( A ) and filter-grown MDCK cells ( B ) were labeled with [ 3 H]cholesterol in the absence (−) or presence (+) of lovastatin/ mevalonate, and radiolabeled cholesterol was allowed to equilibrate with the nonradioactive pool of cholesterol. The cells then were treated for 30 min ( BHK ) or 60 min ( MDCK ) at 37°C with 10 mM methyl-β-cyclodextrin in infection medium (+) or with infection medium only (−). Radioactivity released into the medium and remaining in the cells was determined. Data were obtained from two independent experiments with samples done in duplicate.
    Figure Legend Snippet: Treatment of BHK and MDCK cells with methyl- β-cyclodextrin leads to efficient removal of cellular cholesterol. BHK cells ( A ) and filter-grown MDCK cells ( B ) were labeled with [ 3 H]cholesterol in the absence (−) or presence (+) of lovastatin/ mevalonate, and radiolabeled cholesterol was allowed to equilibrate with the nonradioactive pool of cholesterol. The cells then were treated for 30 min ( BHK ) or 60 min ( MDCK ) at 37°C with 10 mM methyl-β-cyclodextrin in infection medium (+) or with infection medium only (−). Radioactivity released into the medium and remaining in the cells was determined. Data were obtained from two independent experiments with samples done in duplicate.

    Techniques Used: Labeling, Infection, Radioactivity

    20) Product Images from "Stimulatory Effects of Methyl-β-cyclodextrin on Spiramycin Production and Physical–Chemical Characterization of Nonhost@Guest Complexes"

    Article Title: Stimulatory Effects of Methyl-β-cyclodextrin on Spiramycin Production and Physical–Chemical Characterization of Nonhost@Guest Complexes

    Journal: ACS Omega

    doi: 10.1021/acsomega.7b01766

    FT-IR spectra of methyl-β-cyclodextrin (black line), methyl-β-cyclodextrin@spiramycin I complex (gray line), and spiramycin I cyclodextrin (light gray line) in the 1900–800 cm –1 range (A), in the 1800–1650 cm –1 range, (B) and in the 1150–800 cm –1 (C).
    Figure Legend Snippet: FT-IR spectra of methyl-β-cyclodextrin (black line), methyl-β-cyclodextrin@spiramycin I complex (gray line), and spiramycin I cyclodextrin (light gray line) in the 1900–800 cm –1 range (A), in the 1800–1650 cm –1 range, (B) and in the 1150–800 cm –1 (C).

    Techniques Used:

    Complexes between spiramycin I and methyl-β-cyclodextrin as modeled by AutoDock Vina and visualized by Chimera. The images show three different conformations and the relative free energy of the models of complex between spiramycin I and randomly methyled-β-cyclodextrins.
    Figure Legend Snippet: Complexes between spiramycin I and methyl-β-cyclodextrin as modeled by AutoDock Vina and visualized by Chimera. The images show three different conformations and the relative free energy of the models of complex between spiramycin I and randomly methyled-β-cyclodextrins.

    Techniques Used:

    Effects of methyl-β-cyclodextrin on the mycelial growth and spiramycin production by S. ambofaciens . The effects of methyl-β-cyclodextrin (MβCD) on the biomass (A) and total spiramycin production (B) were evaluated during S. ambofaciens growth in the MBM medium containing 25 mM l -valine and 40 g/L glucose. Values indicate mean values ± SD (bars) from quintuplicate experiments. (C). Radar plots showing the percentage ratios between spiramycin I, II, and III produced by S. ambofaciens either in the absence (left) or in the presence of methyl-β-cyclodextrin at 72, 120, and 168 h. Spiramycin production was then evaluated by LC-MS.
    Figure Legend Snippet: Effects of methyl-β-cyclodextrin on the mycelial growth and spiramycin production by S. ambofaciens . The effects of methyl-β-cyclodextrin (MβCD) on the biomass (A) and total spiramycin production (B) were evaluated during S. ambofaciens growth in the MBM medium containing 25 mM l -valine and 40 g/L glucose. Values indicate mean values ± SD (bars) from quintuplicate experiments. (C). Radar plots showing the percentage ratios between spiramycin I, II, and III produced by S. ambofaciens either in the absence (left) or in the presence of methyl-β-cyclodextrin at 72, 120, and 168 h. Spiramycin production was then evaluated by LC-MS.

    Techniques Used: Produced, Liquid Chromatography with Mass Spectroscopy

    (A) UV–vis spectra recorded for the different solutions obtained by mixing specific amounts of spiramycin I and methyl-β-cyclodextrin. A new absorption band located at 279 nm was revealed in the E solution, corresponding to a methyl-β-cyclodextrin/spiramycin I molar ratio of 3:1 (gray line). In the inset a magnification of the 260–310 nm region is reported to better visualize the 279 nm absorption band. (B) Job plot obtained for the complex methyl-β-cyclodextrin@spiramycin I (Δ A at 279 nm and Δ A @279 nm for all acquired spectra vs methyl-β-cyclodextrin molar fraction, χ).
    Figure Legend Snippet: (A) UV–vis spectra recorded for the different solutions obtained by mixing specific amounts of spiramycin I and methyl-β-cyclodextrin. A new absorption band located at 279 nm was revealed in the E solution, corresponding to a methyl-β-cyclodextrin/spiramycin I molar ratio of 3:1 (gray line). In the inset a magnification of the 260–310 nm region is reported to better visualize the 279 nm absorption band. (B) Job plot obtained for the complex methyl-β-cyclodextrin@spiramycin I (Δ A at 279 nm and Δ A @279 nm for all acquired spectra vs methyl-β-cyclodextrin molar fraction, χ).

    Techniques Used:

    21) Product Images from "Neutrophil Extracellular Traps in the Pathogenesis of Equine Recurrent Uveitis (ERU)"

    Article Title: Neutrophil Extracellular Traps in the Pathogenesis of Equine Recurrent Uveitis (ERU)

    Journal: Cells

    doi: 10.3390/cells8121528

    Activation of neutrophils by VBF of horses with healthy eyes and ERU-diseased horses (VBF diseased) and respective nuclease activities (study part II, Hannover). ( a ) After a 240 min incubation period of fresh isolated blood derived neutrophils with VBF, significantly more activated neutrophils were detected in samples from ERU-diseased horses. Roswell Park Memorial Institute medium (RPMI) was used as negative control, with methyl-β-cyclodextrin (CD) diluted in VBF as positive control. The bars represent mean ± SD of five (healthy eyes) or seven (ERU-diseased) independent experiments. Statistical analysis: # represents results of a one-tailed unpaired student’s t -test; * represents results of one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison test. p -values of # p
    Figure Legend Snippet: Activation of neutrophils by VBF of horses with healthy eyes and ERU-diseased horses (VBF diseased) and respective nuclease activities (study part II, Hannover). ( a ) After a 240 min incubation period of fresh isolated blood derived neutrophils with VBF, significantly more activated neutrophils were detected in samples from ERU-diseased horses. Roswell Park Memorial Institute medium (RPMI) was used as negative control, with methyl-β-cyclodextrin (CD) diluted in VBF as positive control. The bars represent mean ± SD of five (healthy eyes) or seven (ERU-diseased) independent experiments. Statistical analysis: # represents results of a one-tailed unpaired student’s t -test; * represents results of one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison test. p -values of # p

    Techniques Used: Activation Assay, Incubation, Isolation, Derivative Assay, Negative Control, Positive Control, One-tailed Test

    22) Product Images from "The Effect of Methyl-β-cyclodextrin on Apoptosis, Proliferative Activity, and Oxidative Stress in Adipose-Derived Mesenchymal Stromal Cells of Horses Suffering from Metabolic Syndrome (EMS)"

    Article Title: The Effect of Methyl-β-cyclodextrin on Apoptosis, Proliferative Activity, and Oxidative Stress in Adipose-Derived Mesenchymal Stromal Cells of Horses Suffering from Metabolic Syndrome (EMS)

    Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

    doi: 10.3390/molecules23020287

    The effect of methyl-β-cyclodextrin on cell morphology. Staining for DAPI, DAPI/phalloidin merged, and MitoRed ( a ). Enlarged cells with oversized nuclei and improper mitochondria distribution are marked on the photographs. The percentage of the enlarged cell area versus the total cell area is presented in ( b ). An asterisks (*) indicates a statistically significant difference in comparison to the control ASC CTRL group. The results expressed as mean ± SD; * p value
    Figure Legend Snippet: The effect of methyl-β-cyclodextrin on cell morphology. Staining for DAPI, DAPI/phalloidin merged, and MitoRed ( a ). Enlarged cells with oversized nuclei and improper mitochondria distribution are marked on the photographs. The percentage of the enlarged cell area versus the total cell area is presented in ( b ). An asterisks (*) indicates a statistically significant difference in comparison to the control ASC CTRL group. The results expressed as mean ± SD; * p value

    Techniques Used: Staining

    23) Product Images from "Endocytosis and intracellular processing of BODIPY-sphingomyelin by murine CATH.a neurons"

    Article Title: Endocytosis and intracellular processing of BODIPY-sphingomyelin by murine CATH.a neurons

    Journal: Biochimica et Biophysica Acta

    doi: 10.1016/j.bbalip.2013.08.007

    Inhibition of BODIPY-SM endocytosis by pharmacological antagonists. To inhibit clathrin-mediated endocytosis, CATH.a cells were incubated in the absence (control) or presence of chlorpromazine (CP; 7.5 μg/ml; 30 min) or were incubated in the presence (+ K + ) or absence (− K + ) of potassium. K + depletion was performed by treatment with K + -free hypotonic buffer (control cells (+ K + ) were treated with hypotonic buffer containing 10 mM KCl). To interfere with caveolar uptake, cells were preincubated in the absence (control) or presence of methyl-β-cyclodextrin (MβCD; 10 mg/ml; 60 min), nystatin (NY; 30 μg/ml; 30 min), genistein (GE; 200 μM; 120 min), or chelerythrine (CHEL; 1.5 μM; 60 min) at 37 °C. Following inhibitor treatment cells were washed with ice-cold HBSS and cooled at 4 °C for 10 min, followed by pulse labeling with BODIPY-SM (2 μM) in serum-free culture medium or the respective K + -free/K + -containing buffers for 30 min at 4 °C. Cells were washed with HBSS and chased for 30 min. After removal of membrane-bound BODIPY-SM by BE cells were subjected to LSM analysis. All incubation steps were performed in the presence of the inhibitors except for MβCD, which was present only during the preincubation period. All micrographs were recorded with the same laser intensity.
    Figure Legend Snippet: Inhibition of BODIPY-SM endocytosis by pharmacological antagonists. To inhibit clathrin-mediated endocytosis, CATH.a cells were incubated in the absence (control) or presence of chlorpromazine (CP; 7.5 μg/ml; 30 min) or were incubated in the presence (+ K + ) or absence (− K + ) of potassium. K + depletion was performed by treatment with K + -free hypotonic buffer (control cells (+ K + ) were treated with hypotonic buffer containing 10 mM KCl). To interfere with caveolar uptake, cells were preincubated in the absence (control) or presence of methyl-β-cyclodextrin (MβCD; 10 mg/ml; 60 min), nystatin (NY; 30 μg/ml; 30 min), genistein (GE; 200 μM; 120 min), or chelerythrine (CHEL; 1.5 μM; 60 min) at 37 °C. Following inhibitor treatment cells were washed with ice-cold HBSS and cooled at 4 °C for 10 min, followed by pulse labeling with BODIPY-SM (2 μM) in serum-free culture medium or the respective K + -free/K + -containing buffers for 30 min at 4 °C. Cells were washed with HBSS and chased for 30 min. After removal of membrane-bound BODIPY-SM by BE cells were subjected to LSM analysis. All incubation steps were performed in the presence of the inhibitors except for MβCD, which was present only during the preincubation period. All micrographs were recorded with the same laser intensity.

    Techniques Used: Inhibition, Incubation, Labeling

    24) Product Images from "Influence of cholesterol/caveolin-1/caveolae homeostasis on membrane properties and substrate adhesion characteristics of adult human mesenchymal stem cells"

    Article Title: Influence of cholesterol/caveolin-1/caveolae homeostasis on membrane properties and substrate adhesion characteristics of adult human mesenchymal stem cells

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/s13287-018-0830-4

    Effects of methyl-β-cyclodextrin (MβCD) and cholesterol (chol)-MβCD treatments on membrane cholesterol level and cell viability of MSCs. a Cholesterol level of MSCs after treatment with MβCD for 40 min at the concentrations indicated. Membrane cholesterol levels were measured and compared (untreated cells = 100%). b Cholesterol level of MSCs after treatment with 10 mM MβCD or 100 μM chol-MβCD for the time indicated. c Time course of cholesterol level of MSCs after initial treatment with 10 mM MβCD for 60 min. After treatment, cells were washed with PBS and incubated in fresh PM containing CS-FBS for the time indicated. d Viability of cells, treated as described in Fig. 1b, evaluated by MTS assay. Results are presented as mean ± SD; n ≥ 3 independent experiments per cell line per time point. The results showed the effectiveness of MβCD and chol-MβCD in altering MSC membrane cholesterol level without compromising cell viability. * p
    Figure Legend Snippet: Effects of methyl-β-cyclodextrin (MβCD) and cholesterol (chol)-MβCD treatments on membrane cholesterol level and cell viability of MSCs. a Cholesterol level of MSCs after treatment with MβCD for 40 min at the concentrations indicated. Membrane cholesterol levels were measured and compared (untreated cells = 100%). b Cholesterol level of MSCs after treatment with 10 mM MβCD or 100 μM chol-MβCD for the time indicated. c Time course of cholesterol level of MSCs after initial treatment with 10 mM MβCD for 60 min. After treatment, cells were washed with PBS and incubated in fresh PM containing CS-FBS for the time indicated. d Viability of cells, treated as described in Fig. 1b, evaluated by MTS assay. Results are presented as mean ± SD; n ≥ 3 independent experiments per cell line per time point. The results showed the effectiveness of MβCD and chol-MβCD in altering MSC membrane cholesterol level without compromising cell viability. * p

    Techniques Used: Incubation, MTS Assay

    25) Product Images from "Cholesterol and F-actin are required for clustering of recycling synaptic vesicle proteins in the presynaptic plasma membrane"

    Article Title: Cholesterol and F-actin are required for clustering of recycling synaptic vesicle proteins in the presynaptic plasma membrane

    Journal: The Journal of Physiology

    doi: 10.1113/jphysiol.2013.265447

    SV proteins are clustered by cholesterol and actin on the plasma membrane during SV recycling. Extraction of vesicular cholesterol by M β CD or actin disruption by LatA causes SV proteins to disperse in the plasma membrane.
    Figure Legend Snippet: SV proteins are clustered by cholesterol and actin on the plasma membrane during SV recycling. Extraction of vesicular cholesterol by M β CD or actin disruption by LatA causes SV proteins to disperse in the plasma membrane.

    Techniques Used:

    A  and  B , schematic diagrams of cholesterol extraction from exocytosed SVs trapped on plasma membrane, where vesicular cholesterol is more accessible to extraction by M β CD and from the plasma membrane of a presynaptic terminal.  C  and  D , live  shi
    Figure Legend Snippet: A and B , schematic diagrams of cholesterol extraction from exocytosed SVs trapped on plasma membrane, where vesicular cholesterol is more accessible to extraction by M β CD and from the plasma membrane of a presynaptic terminal. C and D , live shi

    Techniques Used:

    A  and  B ,  shi  terminals contain numerous AZs (as indicated by the AZ marker brp) at rest and following SV trapping on plasma membrane (10 Hz for 12 min at 30°C) in the presence or absence of 10 m m  M β CD. Note that
    Figure Legend Snippet: A and B , shi terminals contain numerous AZs (as indicated by the AZ marker brp) at rest and following SV trapping on plasma membrane (10 Hz for 12 min at 30°C) in the presence or absence of 10 m m M β CD. Note that

    Techniques Used: Marker

    26) Product Images from "The Endogenous Brain Constituent N-Arachidonoyll-Serine Is an Activator of Large Conductance Ca2+-Activated K+ Channels"

    Article Title: The Endogenous Brain Constituent N-Arachidonoyll-Serine Is an Activator of Large Conductance Ca2+-Activated K+ Channels

    Journal: The Journal of Pharmacology and Experimental Therapeutics

    doi: 10.1124/jpet.108.144717

    Influence of cholesterol manipulation on the effect of ARA-S in HEK293hSlo cell. Cells were preincubated with indicated concentrations of methyl-β-cyclodextrin (β-MCD; depletion) followed by 0.2 mM water-soluble cholesterol (repletion). Control cells were treated with DMEM (for details, see Materials and Methods ). The current was generated by a single ∼90-mV step before ( I time0 ) and after application of 3 μM ARA-S or its vehicle ( I ligand ). The current amplitude before treatment was taken as unity (dashed line). Each bar represents three to nine experiments. ***, P
    Figure Legend Snippet: Influence of cholesterol manipulation on the effect of ARA-S in HEK293hSlo cell. Cells were preincubated with indicated concentrations of methyl-β-cyclodextrin (β-MCD; depletion) followed by 0.2 mM water-soluble cholesterol (repletion). Control cells were treated with DMEM (for details, see Materials and Methods ). The current was generated by a single ∼90-mV step before ( I time0 ) and after application of 3 μM ARA-S or its vehicle ( I ligand ). The current amplitude before treatment was taken as unity (dashed line). Each bar represents three to nine experiments. ***, P

    Techniques Used: Acetylene Reduction Assay, Generated

    27) Product Images from "Inhaled TRIM72 Protein Protects Ventilation Injury to the Lung through Injury-guided Cell Repair"

    Article Title: Inhaled TRIM72 Protein Protects Ventilation Injury to the Lung through Injury-guided Cell Repair

    Journal: American Journal of Respiratory Cell and Molecular Biology

    doi: 10.1165/rcmb.2017-0364OC

    Cholesterol depletion inhibits rhT72 uptake by alveolar epithelial cells. ( A ) Fluorescent images of RFP (RFP-TRIM72 or RFP control, red) and overlay of RFP and FM14-3–labeled plasma membrane (green) in the presence or absence of methyl-β-cyclodextrin (MβCD). Images were taken at 25 minutes after incubation of RLE cells with RFP or RFP-TRIM72 proteins at ×1,000 magnification. Arrows = RLE cells taking up RFP or RFP-TRIM72. ( B ) Quantification of the percentage of RLE cells taking up RFP-tagged protein at 0, 5, 15, and 25 minutes after adding RFP, RFP-T72, or RFP-T72 with MβCD to the cultured cells; n = 3; * P
    Figure Legend Snippet: Cholesterol depletion inhibits rhT72 uptake by alveolar epithelial cells. ( A ) Fluorescent images of RFP (RFP-TRIM72 or RFP control, red) and overlay of RFP and FM14-3–labeled plasma membrane (green) in the presence or absence of methyl-β-cyclodextrin (MβCD). Images were taken at 25 minutes after incubation of RLE cells with RFP or RFP-TRIM72 proteins at ×1,000 magnification. Arrows = RLE cells taking up RFP or RFP-TRIM72. ( B ) Quantification of the percentage of RLE cells taking up RFP-tagged protein at 0, 5, 15, and 25 minutes after adding RFP, RFP-T72, or RFP-T72 with MβCD to the cultured cells; n = 3; * P

    Techniques Used: Labeling, Incubation, Cell Culture

    28) Product Images from "Real-Time Analysis of the Effects of Cholesterol on Lipid Raft Behavior Using Atomic Force Microscopy"

    Article Title: Real-Time Analysis of the Effects of Cholesterol on Lipid Raft Behavior Using Atomic Force Microscopy

    Journal: Biophysical Journal

    doi:

    Effect of cholesterol depletion from a cholesterol-saturated supported bilayer. ( A ) Image of supported lipid bilayer after treatment with water-soluble cholesterol. ( B ) M β CD (20 mM) was injected at the beginning of the scan. The disturbance indicates the point of injection. Initially, a high-cholesterol bilayer was present, exhibiting no lateral heterogeneity. After ∼2 min, discrete rafts were observed, and then, after further cholesterol extraction, the rafts “melted” into the surrounding fluid bilayer. The directions of the scans and the times elapsed at the end of each scan are indicated. Scale bar: 500 nm.
    Figure Legend Snippet: Effect of cholesterol depletion from a cholesterol-saturated supported bilayer. ( A ) Image of supported lipid bilayer after treatment with water-soluble cholesterol. ( B ) M β CD (20 mM) was injected at the beginning of the scan. The disturbance indicates the point of injection. Initially, a high-cholesterol bilayer was present, exhibiting no lateral heterogeneity. After ∼2 min, discrete rafts were observed, and then, after further cholesterol extraction, the rafts “melted” into the surrounding fluid bilayer. The directions of the scans and the times elapsed at the end of each scan are indicated. Scale bar: 500 nm.

    Techniques Used: Injection

    Effect of cholesterol on raft behavior in SM/DOPC monolayers. SM/DOPC monolayers (1:1 mol/mol) containing varying amounts of cholesterol were deposited onto mica from the air-water interface using the Langmuir-Blodgett method at a surface pressure of 30 mN/m. ( A ) No cholesterol, ( B ) 10 mol% cholesterol, ( C ) 20 mol% cholesterol, ( D ) 33% mol cholesterol, ( E ) 50 mol% cholesterol, and ( F ) 50 mol% cholesterol monolayer deposited from a subphase containing 10 mM M β CD. Scale bar: 500 nm.
    Figure Legend Snippet: Effect of cholesterol on raft behavior in SM/DOPC monolayers. SM/DOPC monolayers (1:1 mol/mol) containing varying amounts of cholesterol were deposited onto mica from the air-water interface using the Langmuir-Blodgett method at a surface pressure of 30 mN/m. ( A ) No cholesterol, ( B ) 10 mol% cholesterol, ( C ) 20 mol% cholesterol, ( D ) 33% mol cholesterol, ( E ) 50 mol% cholesterol, and ( F ) 50 mol% cholesterol monolayer deposited from a subphase containing 10 mM M β CD. Scale bar: 500 nm.

    Techniques Used:

    Effect of cholesterol depletion on lipid rafts in supported lipid bilayers. ( A ) SM/DOPC (1:1 mol/mol) bilayer containing 10 mol% cholesterol. Lipid rafts can be clearly seen. ( B ) Addition of M β CD (10 mM) at the beginning of the scan resulted in the loss of lipid raft domains in the bilayer. The disturbance at the bottom of the scan shows the point at which the M β CD was injected. Images  B – D  were captured sequentially by scanning over the same area of the sample. The directions of the scans and the times elapsed at the end of each scan are indicated. Scale bar: 1  μ m.
    Figure Legend Snippet: Effect of cholesterol depletion on lipid rafts in supported lipid bilayers. ( A ) SM/DOPC (1:1 mol/mol) bilayer containing 10 mol% cholesterol. Lipid rafts can be clearly seen. ( B ) Addition of M β CD (10 mM) at the beginning of the scan resulted in the loss of lipid raft domains in the bilayer. The disturbance at the bottom of the scan shows the point at which the M β CD was injected. Images B – D were captured sequentially by scanning over the same area of the sample. The directions of the scans and the times elapsed at the end of each scan are indicated. Scale bar: 1 μ m.

    Techniques Used: Injection

    29) Product Images from "Mechanisms of Alveolar Epithelial Translocation of a Defined Population of Nanoparticles"

    Article Title: Mechanisms of Alveolar Epithelial Translocation of a Defined Population of Nanoparticles

    Journal: American Journal of Respiratory Cell and Molecular Biology

    doi: 10.1165/rcmb.2009-0138OC

    Effects of endocytosis inhibitors on PNP (amidine-modified and carboxylate-modified) flux across the RAECM. PNP flux was measured in the presence and absence of methyl-β-cyclodextrin (caveolin-mediated endocytosis inhibitor; Panel A ( n = 11–13)), chlorpromazine or dansylcadaverine (clathrin-mediated endocytosis inhibitors; Panel B ( n = 7–12)), latrunculin B or cyctochalasin D (phagocytosis and macropinocytosis inhibitors; Panel C ( n = 8–9)), and dynasore (dynamin inhibitor; Panel C ( n = 5)). FITC–cholera toxin B (CTB) (50 μg/ml apical concentration; Panel A ( n = 5)) and Alexa 594–transferrin (500 μg/ml apical concentration; Panel B ( n = 6)) flux across RAECMs was measured as positive control for caveolin- or clathrin-mediated endocytosis, respectively. *Significantly less than control; † significantly greater than control.
    Figure Legend Snippet: Effects of endocytosis inhibitors on PNP (amidine-modified and carboxylate-modified) flux across the RAECM. PNP flux was measured in the presence and absence of methyl-β-cyclodextrin (caveolin-mediated endocytosis inhibitor; Panel A ( n = 11–13)), chlorpromazine or dansylcadaverine (clathrin-mediated endocytosis inhibitors; Panel B ( n = 7–12)), latrunculin B or cyctochalasin D (phagocytosis and macropinocytosis inhibitors; Panel C ( n = 8–9)), and dynasore (dynamin inhibitor; Panel C ( n = 5)). FITC–cholera toxin B (CTB) (50 μg/ml apical concentration; Panel A ( n = 5)) and Alexa 594–transferrin (500 μg/ml apical concentration; Panel B ( n = 6)) flux across RAECMs was measured as positive control for caveolin- or clathrin-mediated endocytosis, respectively. *Significantly less than control; † significantly greater than control.

    Techniques Used: Modification, CtB Assay, Concentration Assay, Positive Control

    30) Product Images from "Drug-Eluting Fibers for HIV-1 Inhibition and Contraception"

    Article Title: Drug-Eluting Fibers for HIV-1 Inhibition and Contraception

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0049792

    Electrospun fibers incorporate drugs for multipurpose prevention. ( a ) Two-axis mandrel electrospinning rig for fiber collection. ( b ) Controlled fiber deposition along a grounded aluminum collector produces a geometry that may be suitable for vaginal drug delivery. ( c ) Mesh abstracted from mandrel has a hollow interior. ( d ) Fiber meshes have porous microstructure. ( e ) Combining fiber meshes produces a multifunctional material. ( f ) Diverse agents with action against HIV, HSV-2, or sperm are incorporated into blends of PLLA and PEO. PLLA/PEO (30∶70, blue) and PLLA/PEO (70∶30, red); AZT = 1 wt% 3′-azido-3′-deoxythymidine, MVC = 1 wt% maraviroc, ACV = 1 wt% acycloguanosine, GML = 10 wt% glycerol monolaurate, MBCD = 10 wt% methyl-β-cyclodextrin, Fe/Asc = 10 wt% iron (II) D-gluconate with 10 wt% ascorbic acid.
    Figure Legend Snippet: Electrospun fibers incorporate drugs for multipurpose prevention. ( a ) Two-axis mandrel electrospinning rig for fiber collection. ( b ) Controlled fiber deposition along a grounded aluminum collector produces a geometry that may be suitable for vaginal drug delivery. ( c ) Mesh abstracted from mandrel has a hollow interior. ( d ) Fiber meshes have porous microstructure. ( e ) Combining fiber meshes produces a multifunctional material. ( f ) Diverse agents with action against HIV, HSV-2, or sperm are incorporated into blends of PLLA and PEO. PLLA/PEO (30∶70, blue) and PLLA/PEO (70∶30, red); AZT = 1 wt% 3′-azido-3′-deoxythymidine, MVC = 1 wt% maraviroc, ACV = 1 wt% acycloguanosine, GML = 10 wt% glycerol monolaurate, MBCD = 10 wt% methyl-β-cyclodextrin, Fe/Asc = 10 wt% iron (II) D-gluconate with 10 wt% ascorbic acid.

    Techniques Used:

    31) Product Images from "Cholesterol trafficking and raft-like membrane domain composition mediate scavenger receptor class B type 1-dependent lipid sensing in intestinal epithelial cells"

    Article Title: Cholesterol trafficking and raft-like membrane domain composition mediate scavenger receptor class B type 1-dependent lipid sensing in intestinal epithelial cells

    Journal: Biochimica et biophysica acta

    doi: 10.1016/j.bbalip.2017.11.009

    SR-B1 is involved in cholesterol trafficking from plasma membrane ( A ) Pulse-chase experiments were conducted on Caco-2/TC7 cells stably transfected with WT SR-B1 or SR-B1-Q445A. Cells were cultured on semi-permeable filters and incubated in presence of BODIPY-cholesterol combined with methyl- β-cyclodextrin (Bdp-Chol/CD) in the apical compartment for 1 min (pulse), and then with culture medium without cholesterol for 5 min and 1 hour (chase). BODIPY-Cholesterol (purple) distribution was analyzed by confocal microscopy. DAPI (blue) is used for visualization of nucleus. Sub-apical XY planes are shown. Bar= 10μm. ( B ) Summary data (mean ± SEM) for Bdp-Chol fluorescence intensity quantified at sub-apical XY planes (5 fields per condition) are expressed as arbitrary units (a.u.). **p
    Figure Legend Snippet: SR-B1 is involved in cholesterol trafficking from plasma membrane ( A ) Pulse-chase experiments were conducted on Caco-2/TC7 cells stably transfected with WT SR-B1 or SR-B1-Q445A. Cells were cultured on semi-permeable filters and incubated in presence of BODIPY-cholesterol combined with methyl- β-cyclodextrin (Bdp-Chol/CD) in the apical compartment for 1 min (pulse), and then with culture medium without cholesterol for 5 min and 1 hour (chase). BODIPY-Cholesterol (purple) distribution was analyzed by confocal microscopy. DAPI (blue) is used for visualization of nucleus. Sub-apical XY planes are shown. Bar= 10μm. ( B ) Summary data (mean ± SEM) for Bdp-Chol fluorescence intensity quantified at sub-apical XY planes (5 fields per condition) are expressed as arbitrary units (a.u.). **p

    Techniques Used: Pulse Chase, Stable Transfection, Transfection, Cell Culture, Incubation, Confocal Microscopy, Fluorescence

    32) Product Images from "Counteracting Signaling Activities in Lipid Rafts Associated with the Invasion of Lung Epithelial Cells by Pseudomonas aeruginosa"

    Article Title: Counteracting Signaling Activities in Lipid Rafts Associated with the Invasion of Lung Epithelial Cells by Pseudomonas aeruginosa

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M808629200

    Tyrosine phosphorylation of caveolin-2 regulates the lipid raft-mediated entry of Pa. A , the increased uptake of Pa in pFL-cav-2 cells was inhibited with methyl-β-cyclodextrin ( p = 0.004) and genistein ( p = 0.021), indicating the dependence
    Figure Legend Snippet: Tyrosine phosphorylation of caveolin-2 regulates the lipid raft-mediated entry of Pa. A , the increased uptake of Pa in pFL-cav-2 cells was inhibited with methyl-β-cyclodextrin ( p = 0.004) and genistein ( p = 0.021), indicating the dependence

    Techniques Used:

    33) Product Images from "Hexokinase II–derived cell-penetrating peptide targets mitochondria and triggers apoptosis in cancer cells"

    Article Title: Hexokinase II–derived cell-penetrating peptide targets mitochondria and triggers apoptosis in cancer cells

    Journal: The FASEB Journal

    doi: 10.1096/fj.201601173R

    Determination of cellular uptake mechanisms of pHK and pHK-PAS. A ) Effects of low temperature and energy depletion on peptide internalization. HeLa cells were preincubated for 1 h at 4°C in serum-free DMEM or pretreated for 1 h at 37°C with 10 mM sodium azide and 6 mM 2-deoxy- d -glucose in serum- and glucose-free DMEM to deplete cellular ATP. pHK A488 (25 µM; left) or pHK-PAS A488 (25 µM; middle) was then added, and cells were maintained for 2 h at 4°C or in the presence of sodium azide/2-deoxy- d -glucose at 37°C. B ) Effects of endocytosis inhibitors on peptide internalization. HeLa cells were treated for 30 min at 37°C in serum-free DMEM with the following: 10 µM chlorpromazine (Chlor; clathrin-dependent endocytosis), 5 mM methyl-β-cyclodextrin (MβCD; lipid raft–mediated endocytosis), 4 µM filipin (Filip; caveolae-dependent endocytosis), 10 µM nocodazole (Nocod; microtubule polymerization), or 10 µM cytochalasin D (Cyto D; macropinocytosis). Cells were then treated with 25 µM pHK A488 (left) or pHK-PAS A488 (middle) and maintained for 2 h at 37°C in the presence of inhibitors and peptides. Thereafter, cells were washed 3 times with ice-cold PBS, trypsinized, centrifuged, and resuspended in ice-cold PBS with 10% FBS, and fluorescence was measured by FACS. Cells that were treated with peptide without inhibitors at 37°C were used as control, and cells that were treated with vehicle alone served as background. Uptake efficiencies (right) were determined from the ratio of fluorescence of cells treated with peptide under different inhibition conditions to control cells. ns, nonsignificant ( P > 0.05). ** P
    Figure Legend Snippet: Determination of cellular uptake mechanisms of pHK and pHK-PAS. A ) Effects of low temperature and energy depletion on peptide internalization. HeLa cells were preincubated for 1 h at 4°C in serum-free DMEM or pretreated for 1 h at 37°C with 10 mM sodium azide and 6 mM 2-deoxy- d -glucose in serum- and glucose-free DMEM to deplete cellular ATP. pHK A488 (25 µM; left) or pHK-PAS A488 (25 µM; middle) was then added, and cells were maintained for 2 h at 4°C or in the presence of sodium azide/2-deoxy- d -glucose at 37°C. B ) Effects of endocytosis inhibitors on peptide internalization. HeLa cells were treated for 30 min at 37°C in serum-free DMEM with the following: 10 µM chlorpromazine (Chlor; clathrin-dependent endocytosis), 5 mM methyl-β-cyclodextrin (MβCD; lipid raft–mediated endocytosis), 4 µM filipin (Filip; caveolae-dependent endocytosis), 10 µM nocodazole (Nocod; microtubule polymerization), or 10 µM cytochalasin D (Cyto D; macropinocytosis). Cells were then treated with 25 µM pHK A488 (left) or pHK-PAS A488 (middle) and maintained for 2 h at 37°C in the presence of inhibitors and peptides. Thereafter, cells were washed 3 times with ice-cold PBS, trypsinized, centrifuged, and resuspended in ice-cold PBS with 10% FBS, and fluorescence was measured by FACS. Cells that were treated with peptide without inhibitors at 37°C were used as control, and cells that were treated with vehicle alone served as background. Uptake efficiencies (right) were determined from the ratio of fluorescence of cells treated with peptide under different inhibition conditions to control cells. ns, nonsignificant ( P > 0.05). ** P

    Techniques Used: Fluorescence, FACS, Inhibition

    34) Product Images from "Hypoxia regulates global membrane protein endocytosis through caveolin-1 in cancer cells"

    Article Title: Hypoxia regulates global membrane protein endocytosis through caveolin-1 in cancer cells

    Journal: Nature Communications

    doi: 10.1038/ncomms11371

    Dynamics of constitutive membrane protein endocytosis. ( a ) Schematic outline of procedures for the quantification and encoding of cell-surface proteome endocytosis, as described in Methods. ( b ) Representative confocal microscopy images of cell-surface and internalized biotinylated proteins in HeLa cells at the indicated time points (see also, Supplementary Movie 1 ). Scale bar, 20 μm. ( c ) FACS quantification of biotinylated cell-surface, and endocytosed membrane proteome in HeLa cells following 30 min of internalization; left panel: representative histograms of non-biotinylated cells, total cell-surface biotinylation, residual cell-surface signal following reductive cleavage of biotin-protein linker with MesNa and internalized membrane proteins; right panel: quantitative analysis presented as the mean±s.d. from three independent experiments each performed in duplicates. MFI, median fluorescence intensity. ( d ) Representative confocal microscopy images of HeLa cells from experiment described in ( c ). Scale bar, 20 μm; lower right panel, 10 μm. ( e ) Time-dependent internalization in HeLa cells of cell-surface proteome presented as % of total cell-surface biotinylation at t =0, that is, without induction of endocytosis. ( f ) Confocal microscopy imaging of biotinylated proteins (magenta) and the early endosome marker EEA1 (green) in HeLa cells shows co-localization following 30 min of endocytosis. Arrows in lower right panel indicate co-localization. Shown are representative images from at least 3 independent experiments. Scale bar, 10 μm; lower right panel, 3.5 μm. ( g–k ) FACS quantification of constitutive biotinylated membrane protein endocytosis at 30 min following pre-treatment of HeLa cells with ( g ) dynamin inhibitor Dynasore for 30 min, ( h ) membrane cholesterol depletion agent methyl-β-cyclodextrin (MCD) for 30 min, ( i ) low-density lipoprotein cholesterol loading for 20 h, ( j ) macropinocytosis inhibitor wortmannin for 1 h and ( k ) ERK1/2 phosphorylation inhibitor UO126 for 1 h at the indicated concentrations. Data are presented as % of untreated control cells (Ctrl)±s.d. from a representative experiment. NS, not significant. * P
    Figure Legend Snippet: Dynamics of constitutive membrane protein endocytosis. ( a ) Schematic outline of procedures for the quantification and encoding of cell-surface proteome endocytosis, as described in Methods. ( b ) Representative confocal microscopy images of cell-surface and internalized biotinylated proteins in HeLa cells at the indicated time points (see also, Supplementary Movie 1 ). Scale bar, 20 μm. ( c ) FACS quantification of biotinylated cell-surface, and endocytosed membrane proteome in HeLa cells following 30 min of internalization; left panel: representative histograms of non-biotinylated cells, total cell-surface biotinylation, residual cell-surface signal following reductive cleavage of biotin-protein linker with MesNa and internalized membrane proteins; right panel: quantitative analysis presented as the mean±s.d. from three independent experiments each performed in duplicates. MFI, median fluorescence intensity. ( d ) Representative confocal microscopy images of HeLa cells from experiment described in ( c ). Scale bar, 20 μm; lower right panel, 10 μm. ( e ) Time-dependent internalization in HeLa cells of cell-surface proteome presented as % of total cell-surface biotinylation at t =0, that is, without induction of endocytosis. ( f ) Confocal microscopy imaging of biotinylated proteins (magenta) and the early endosome marker EEA1 (green) in HeLa cells shows co-localization following 30 min of endocytosis. Arrows in lower right panel indicate co-localization. Shown are representative images from at least 3 independent experiments. Scale bar, 10 μm; lower right panel, 3.5 μm. ( g–k ) FACS quantification of constitutive biotinylated membrane protein endocytosis at 30 min following pre-treatment of HeLa cells with ( g ) dynamin inhibitor Dynasore for 30 min, ( h ) membrane cholesterol depletion agent methyl-β-cyclodextrin (MCD) for 30 min, ( i ) low-density lipoprotein cholesterol loading for 20 h, ( j ) macropinocytosis inhibitor wortmannin for 1 h and ( k ) ERK1/2 phosphorylation inhibitor UO126 for 1 h at the indicated concentrations. Data are presented as % of untreated control cells (Ctrl)±s.d. from a representative experiment. NS, not significant. * P

    Techniques Used: Confocal Microscopy, FACS, Fluorescence, Imaging, Marker

    35) Product Images from "Adenylate Cyclase Toxin promotes bacterial internalisation into non phagocytic cells"

    Article Title: Adenylate Cyclase Toxin promotes bacterial internalisation into non phagocytic cells

    Journal: Scientific Reports

    doi: 10.1038/srep13774

    Characterization of the invasion pathway of “ACT-coated bacteria”. Several inhibitors of the different possible entry routes were analyzed and it was concluded that “ACT-coated B. pertussis” exploits a clathrin-independent, cholesterol-dependent (caveolae-dependent) entry pathway to invade CHO-K1 cells, and that it requires the activation of tyrosine kinases. CHO-K1 cells were pre-incubated with chemical inhibitors (5 μg/mL chlorpromazine, 450 mM sucrose, 10 mM methyl-β-cyclodextrin, 0.25 μg/mL nystatin, 100 μM genistein and 1 μM okadaic acid) for 30 min at 37 °C, then cell invasion was assayed as described in Experimental procedures. Chlorpromazine, sucrose and methyl-β-cyclodextrin were dissolved in DMEM, nystatin, genistein and okadaic acid, were dissolved in DMSO. Controls showing that vehicle has no effect on internalisation are shown in Fig. S5. The data were normalized to the control sample ( no addition ) and expressed as per cent of control entry. Data shown are the mean ± SD of at least three independent experiments performed in quintuplicate, with *p
    Figure Legend Snippet: Characterization of the invasion pathway of “ACT-coated bacteria”. Several inhibitors of the different possible entry routes were analyzed and it was concluded that “ACT-coated B. pertussis” exploits a clathrin-independent, cholesterol-dependent (caveolae-dependent) entry pathway to invade CHO-K1 cells, and that it requires the activation of tyrosine kinases. CHO-K1 cells were pre-incubated with chemical inhibitors (5 μg/mL chlorpromazine, 450 mM sucrose, 10 mM methyl-β-cyclodextrin, 0.25 μg/mL nystatin, 100 μM genistein and 1 μM okadaic acid) for 30 min at 37 °C, then cell invasion was assayed as described in Experimental procedures. Chlorpromazine, sucrose and methyl-β-cyclodextrin were dissolved in DMEM, nystatin, genistein and okadaic acid, were dissolved in DMSO. Controls showing that vehicle has no effect on internalisation are shown in Fig. S5. The data were normalized to the control sample ( no addition ) and expressed as per cent of control entry. Data shown are the mean ± SD of at least three independent experiments performed in quintuplicate, with *p

    Techniques Used: Activation Assay, Incubation

    36) Product Images from "Rapid activation of monocyte tissue factor by antithymocyte globulin is dependent on complement and protein disulfide isomerase"

    Article Title: Rapid activation of monocyte tissue factor by antithymocyte globulin is dependent on complement and protein disulfide isomerase

    Journal: Blood

    doi: 10.1182/blood-2012-10-460493

    TF activation by ATG requires lipid raft integrity, but does not involve TFPI inhibition. (A) THP1 cells were incubated with PBS, IgG, or ATG (100 µg/mL), washed, and either left intact or disrupted by repeated freeze-thawing. Intact and disrupted cells were mixed 1:1 (v:v) with NHP for 5 minutes at 37°C and analyzed for PCA by a single-stage clotting assay (mean ± SD, n = 3). THP1 cells were pretreated with (B) filipin or (C) methyl-β-cyclodextrin (MBCD) before being loaded with rabbit IgG or ATG (100 µg/mL) and analyzed for PCA as described above (mean ± SD, n = 5). (D) Binding of IgG or ATG (100 µg/mL) to THP1 cells was evaluated using indirect flow cytometry with or without (left) filipin or (right) MBCD pretreatment. Representative histograms are shown. (E) Analysis of TFPI antigen expression on THP1 cells by indirect flow cytometry using polyclonal anti-TFPI (αTFPI) in comparison with control IgG. A representative experiment is shown. (F) THP1 cells were suspended in calcium-HBS and incubated with PBS, ATG (100 µg/mL), inhibitory anti-TFPI (50 µg/mL), and/or calcium ionophore A23187 (20 µM) for 15 minutes at 37°C. Cells were washed, resuspended in PBS, and exposed to NHP before PCA was measured as described above (mean ± SD, n = 3).
    Figure Legend Snippet: TF activation by ATG requires lipid raft integrity, but does not involve TFPI inhibition. (A) THP1 cells were incubated with PBS, IgG, or ATG (100 µg/mL), washed, and either left intact or disrupted by repeated freeze-thawing. Intact and disrupted cells were mixed 1:1 (v:v) with NHP for 5 minutes at 37°C and analyzed for PCA by a single-stage clotting assay (mean ± SD, n = 3). THP1 cells were pretreated with (B) filipin or (C) methyl-β-cyclodextrin (MBCD) before being loaded with rabbit IgG or ATG (100 µg/mL) and analyzed for PCA as described above (mean ± SD, n = 5). (D) Binding of IgG or ATG (100 µg/mL) to THP1 cells was evaluated using indirect flow cytometry with or without (left) filipin or (right) MBCD pretreatment. Representative histograms are shown. (E) Analysis of TFPI antigen expression on THP1 cells by indirect flow cytometry using polyclonal anti-TFPI (αTFPI) in comparison with control IgG. A representative experiment is shown. (F) THP1 cells were suspended in calcium-HBS and incubated with PBS, ATG (100 µg/mL), inhibitory anti-TFPI (50 µg/mL), and/or calcium ionophore A23187 (20 µM) for 15 minutes at 37°C. Cells were washed, resuspended in PBS, and exposed to NHP before PCA was measured as described above (mean ± SD, n = 3).

    Techniques Used: Activation Assay, Inhibition, Incubation, Coagulation, Binding Assay, Flow Cytometry, Cytometry, Expressing

    37) Product Images from "Synergistic enhancement of production of proinflammatory cytokines of human peripheral blood monocytes by anti-Sm and anti-RNP antibodies"

    Article Title: Synergistic enhancement of production of proinflammatory cytokines of human peripheral blood monocytes by anti-Sm and anti-RNP antibodies

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0209282

    Effects of methyl-β-cyclodextrin and cytochalasin D on the synergistic enhancement of the production of IL-6 of peripheral blood monocytes by anti-Sm mAb and anti-RNP mAb. Highly purified monocytes were cultured in the presence or absence of methyl-β-cyclodextrin (5mM) and cytochalasin D (10 μM) with various combination of anti-Sm mAb, anti-RNP mAb, control IgG1 or IgG3 (3 μg/ml). After 48 hours of incubation, the supernatants were assayed for IL-6. Mean values with standard deviation (error bars) of 2 different experiments with reproducible results are shown.
    Figure Legend Snippet: Effects of methyl-β-cyclodextrin and cytochalasin D on the synergistic enhancement of the production of IL-6 of peripheral blood monocytes by anti-Sm mAb and anti-RNP mAb. Highly purified monocytes were cultured in the presence or absence of methyl-β-cyclodextrin (5mM) and cytochalasin D (10 μM) with various combination of anti-Sm mAb, anti-RNP mAb, control IgG1 or IgG3 (3 μg/ml). After 48 hours of incubation, the supernatants were assayed for IL-6. Mean values with standard deviation (error bars) of 2 different experiments with reproducible results are shown.

    Techniques Used: Purification, Cell Culture, Incubation, Standard Deviation

    38) Product Images from "Overexpression of OSBP-related protein 2 (ORP2) induces changes in cellular cholesterol metabolism and enhances endocytosis"

    Article Title: Overexpression of OSBP-related protein 2 (ORP2) induces changes in cellular cholesterol metabolism and enhances endocytosis

    Journal:

    doi: 10.1042/BJ20042082

    Effect of ORP2 overexpression on the efflux of newly synthesized cholesterol to methyl-β-cyclodextrin
    Figure Legend Snippet: Effect of ORP2 overexpression on the efflux of newly synthesized cholesterol to methyl-β-cyclodextrin

    Techniques Used: Over Expression, Synthesized

    39) Product Images from "Characterization of the ?1-Adrenoceptor Subtype Activating Extracellular Signal-Regulated Kinase in Submandibular Gland Acinar Cells"

    Article Title: Characterization of the ?1-Adrenoceptor Subtype Activating Extracellular Signal-Regulated Kinase in Submandibular Gland Acinar Cells

    Journal:

    doi: 10.1016/j.ejphar.2007.09.029

    Subcellular location of α 1 -adrenoceptors and effects of cholesterol depletion/disruption by filipin (Fil) or methyl-β-cyclodextrin (mβCD) on phenylephrine (PE) stimulated ERK1/2 in SMG-C10 cells. (A) Sucrose density gradient fractionation
    Figure Legend Snippet: Subcellular location of α 1 -adrenoceptors and effects of cholesterol depletion/disruption by filipin (Fil) or methyl-β-cyclodextrin (mβCD) on phenylephrine (PE) stimulated ERK1/2 in SMG-C10 cells. (A) Sucrose density gradient fractionation

    Techniques Used: Fractionation

    40) Product Images from "Human Cytomegalovirus Entry into Dendritic Cells Occurs via a Macropinocytosis-Like Pathway in a pH-Independent and Cholesterol-Dependent Manner"

    Article Title: Human Cytomegalovirus Entry into Dendritic Cells Occurs via a Macropinocytosis-Like Pathway in a pH-Independent and Cholesterol-Dependent Manner

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0034795

    Cholesterol depletion is detrimental to the HCMV entry into MDDCs. A) Cells were pre-incubated with filipin (7.66, 1.5, 0.3 µM), nystatin (21.2, 4.3, 0.85 µM) or methyl-β-cyclodextrin (MβCD; 5, 1, 0.2 mM) or with vehicle (DMSO, 1/100) and were processed as described in the legend for Figure 1D . For nystatin, n= 2 independent experiments with 2 different donors in total; for Filipin and MβCD n=4 independent experiments with 6 different donors in total. ns : not significant (p=0,0535).
    Figure Legend Snippet: Cholesterol depletion is detrimental to the HCMV entry into MDDCs. A) Cells were pre-incubated with filipin (7.66, 1.5, 0.3 µM), nystatin (21.2, 4.3, 0.85 µM) or methyl-β-cyclodextrin (MβCD; 5, 1, 0.2 mM) or with vehicle (DMSO, 1/100) and were processed as described in the legend for Figure 1D . For nystatin, n= 2 independent experiments with 2 different donors in total; for Filipin and MβCD n=4 independent experiments with 6 different donors in total. ns : not significant (p=0,0535).

    Techniques Used: Incubation

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    Protease Inhibitor:

    Article Title: Lipid Raft-dependent Glucagon-like Peptide-2 Receptor Trafficking Occurs Independently of Agonist-induced Desensitization
    Article Snippet: .. Forskolin, 3-isobutyl-1-methylxanthine (IBMX), forskolin, anti-FLAG (M1) antibody, anti-mouse IgG-agarose, mouse IgG, 2,2′-azino-bis(3-ethylbenthiazoline-6-sulfonic acid) reagent, protease inhibitor cocktail, phosphatase inhibitor cocktail I, filipin complex, methyl-β-cyclodextrin (MβCD), and sodium monensin were all obtained from Sigma-Aldrich (St. Louis, MO). .. Peptide N -glycanase F (PNGase F) and restriction enzymes were from New England Biolabs (Mississauga, Ontario, Canada).

    Blocking Assay:

    Article Title: Modulation of deregulated chaperone-mediated autophagy by a phosphopeptide
    Article Snippet: .. Ten μg/mL CPZ (Sigma-Aldrich, C-8138) was used to inhibit clathrin-mediated endocytosis, 5 μg/mL filipin (Sigma-Aldrich, F-4767) to disrupt caveolae-dependent endocytosis, and 5 mM methyl-β-cyclodextrin (Sigma-Aldrich, C-4555) to block macropinocytosis. .. The inhibitory effect of the compounds was verified with well-known fluorescent substrates for each endocytosis pathway, namely 100 μg/mL TF/transferrin (Life technologies, T13342) for CPZ, 0.5 μM BODIPY FL C5- Lactosylceramide/bovine serum albumin (Life Technologies, I34402) for filipin and 250 μg/mL dextran (Life Technologies, D1820) for methyl-β-cyclodextrin.

    Incubation:

    Article Title: High-resolution imaging and quantification of plasma membrane cholesterol by NanoSIMS
    Article Snippet: .. To deplete cholesterol, cells were incubated with 10 mM methyl-β-cyclodextrin (MβCD, Sigma) in Ham’s F-12 Nutrient Mixture at 37 °C for 15 min. To release the sphingomyelin-sequestered pool of cholesterol on the plasma membrane, cells were incubated at 37 °C for 30 min in medium A containing 100 milliunits/mL of sphingomyelinase from Staphylococcus aureus (Sigma). ..

    Article Title: Inhibiting mevalonate pathway enzymes increases stromal cell resilience to a cholesterol-dependent cytolysin
    Article Snippet: .. To examine the potential binding to PLO, 1 μM atorvastatin, 10 μM alendronate, 10 μM zaragozic acid, 1 mM methyl-β-cyclodextrin, and 1 mM cholesterol (all Sigma) were incubated with 100 HU/ml PLO for 1 h, prior to conducting the hemolysis assay. .. Cell culture Isolation and culture of primary bovine endometrial stromal cells was performed as described previously , .

    other:

    Article Title: Complexation of C6-Ceramide with Cholesteryl Phosphocholine - A Potent Solvent-Free Ceramide Delivery Formulation for Cells in Culture
    Article Snippet: Materials Cholesterol, methyl-β-cyclodextrin, and dimethyl sulfoxide (DMSO) were obtained from Sigma/Aldrich (St. Louis, MO).

    Cell Culture:

    Article Title: Plasma membrane order and fluidity are diversely triggered by elicitors of plant defence
    Article Snippet: .. Reagents 1-[2-Hydroxy-3-(N ,N -di-methyl-N -hydroxyethyl)ammoniopropyl]-4-[β-[2-(di-n -butylamino)-6-napthyl]vinyl] pyridinium dibromide (di-4-ANEPPDHQ) was purchased from Molecular Probes Inc. Staurosporin, lanthanum (La3+ ), diphenyleneiodonium (DPI), and methyl-β-cyclodextrin (MβCD; Cell Culture Tested) were obtained from Sigma-Aldrich. .. Hydrogen peroxide (H2 O2 , 3 wt % solution in water containing 200ppm acetanilide as stabilizer) was also supplied by Sigma-Aldrich.

    Binding Assay:

    Article Title: Inhibiting mevalonate pathway enzymes increases stromal cell resilience to a cholesterol-dependent cytolysin
    Article Snippet: .. To examine the potential binding to PLO, 1 μM atorvastatin, 10 μM alendronate, 10 μM zaragozic acid, 1 mM methyl-β-cyclodextrin, and 1 mM cholesterol (all Sigma) were incubated with 100 HU/ml PLO for 1 h, prior to conducting the hemolysis assay. .. Cell culture Isolation and culture of primary bovine endometrial stromal cells was performed as described previously , .

    Hemolysis Assay:

    Article Title: Inhibiting mevalonate pathway enzymes increases stromal cell resilience to a cholesterol-dependent cytolysin
    Article Snippet: .. To examine the potential binding to PLO, 1 μM atorvastatin, 10 μM alendronate, 10 μM zaragozic acid, 1 mM methyl-β-cyclodextrin, and 1 mM cholesterol (all Sigma) were incubated with 100 HU/ml PLO for 1 h, prior to conducting the hemolysis assay. .. Cell culture Isolation and culture of primary bovine endometrial stromal cells was performed as described previously , .

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    Millipore cytochrome c reduction
    Superoxide reductase activity of FA796 in a <t>cytochrome</t> c assay. The assay was performed in 96-well plate under aerobic conditions, in a 100 µl total reaction volume in a buffer containing 100 mM Tris.HCl and 150 mM NaCl, pH 8.0. The reactions were followed by measuring absorbance at 550 nm for 10 minutes with 1 minute interval. ( A ) Cytochrome c (20 µM) was reduced in aerobic solutions resulting in an increased absorbance at 550 nm. ( B ) Generation of superoxide radicals by addition of xanthine (0.2 mM) and xanthine oxidase (0.0005 units) to the reaction mixture strongly enhanced the reduction of cytochrome c. ( C ) Addition of r-FA796 (1 µM) to the reaction mixture inhibited the reduction of cytochrome c by competing for the superoxide radicals. ( D ) Addition of 5.3 µM FA796 protein completely prevented the reduction of cytochrome c. ( E ) Addition of excess FA796 (10 µM) caused oxidation of the reduced cytochrome c. The results represent the means of three independent experiments. Error bars represent the standard deviations from the means.
    Cytochrome C Reduction, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore meβcd
    Cortical 45-kDa Gαs is contained in low-density membrane and is reduced after treatment of oocytes with progesterone or <t>MeβCD</t>
    Meβcd, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore mβcd powder
    Recovery of dysregulated protein signature in NPC1 fibroblasts upon <t>MβCD</t> treatment. (A) Heat map showing the identified and quantified proteins that are differentially expressed in NPC1 fibroblast cells, comparing with WT, and reversed by MβCD-1, MβCD-2, or MβCD-3 treatments. The color key indicates the relative abundance of proteins (0–1.0) across five samples. Relative protein levels of NPC2 (B) , USE1 (C) , VAMP7 (D) , GABARAP (E) , NSDHL (F) , and DHCR24 (G) . CD, cyclodextrin; ppm, part per million.
    Mβcd Powder, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Superoxide reductase activity of FA796 in a cytochrome c assay. The assay was performed in 96-well plate under aerobic conditions, in a 100 µl total reaction volume in a buffer containing 100 mM Tris.HCl and 150 mM NaCl, pH 8.0. The reactions were followed by measuring absorbance at 550 nm for 10 minutes with 1 minute interval. ( A ) Cytochrome c (20 µM) was reduced in aerobic solutions resulting in an increased absorbance at 550 nm. ( B ) Generation of superoxide radicals by addition of xanthine (0.2 mM) and xanthine oxidase (0.0005 units) to the reaction mixture strongly enhanced the reduction of cytochrome c. ( C ) Addition of r-FA796 (1 µM) to the reaction mixture inhibited the reduction of cytochrome c by competing for the superoxide radicals. ( D ) Addition of 5.3 µM FA796 protein completely prevented the reduction of cytochrome c. ( E ) Addition of excess FA796 (10 µM) caused oxidation of the reduced cytochrome c. The results represent the means of three independent experiments. Error bars represent the standard deviations from the means.

    Journal: Scientific Reports

    Article Title: Role of Superoxide Reductase FA796 in Oxidative Stress Resistance in Filifactor alocis

    doi: 10.1038/s41598-020-65806-3

    Figure Lengend Snippet: Superoxide reductase activity of FA796 in a cytochrome c assay. The assay was performed in 96-well plate under aerobic conditions, in a 100 µl total reaction volume in a buffer containing 100 mM Tris.HCl and 150 mM NaCl, pH 8.0. The reactions were followed by measuring absorbance at 550 nm for 10 minutes with 1 minute interval. ( A ) Cytochrome c (20 µM) was reduced in aerobic solutions resulting in an increased absorbance at 550 nm. ( B ) Generation of superoxide radicals by addition of xanthine (0.2 mM) and xanthine oxidase (0.0005 units) to the reaction mixture strongly enhanced the reduction of cytochrome c. ( C ) Addition of r-FA796 (1 µM) to the reaction mixture inhibited the reduction of cytochrome c by competing for the superoxide radicals. ( D ) Addition of 5.3 µM FA796 protein completely prevented the reduction of cytochrome c. ( E ) Addition of excess FA796 (10 µM) caused oxidation of the reduced cytochrome c. The results represent the means of three independent experiments. Error bars represent the standard deviations from the means.

    Article Snippet: Superoxide reductase activity assay The ability of r-FA796 to catalyze the reduction of superoxide radical was determined by monitoring the inhibition of cytochrome c reduction by superoxide radicals generated from xanthine/xanthine oxidase reaction .

    Techniques: Activity Assay

    Functional expression of NOX1 isoforms in human colon cancer cell lines. (A) RT-PCR and subsequent DNA gel analyses were performed to detect NOX1-L and NOX1-S / NOX1-Lv in a subset of 8 colon cancer cell lines plus HEK293-NOX1. (B) The subset of cell lines was evaluated for PMA-stimulated superoxide production by luminescence assay. (C-G) Basal and PMA-stimulated superoxide production were also measured in (C) HEK293-vector control, (D) HEK293-NOX1, (E) LS513, (F) HT-29, and (G) RKO cells using the luminol assay. Superoxide dismutase-polyethylene glycol (PEG-SOD, 200 U/ml) was used to confirm superoxide production, with measurements taken every 2 min for up to 120 min. (H) Mean rate of superoxide production at 60 min in nmol/h/10 6 cells. 2X10 6 cells per well were suspended in 200 μl of HBSS-HEPES containing 100 μM acetylated cytochrome c with or without 200 nM PMA and/or 200 U/ml PEG-SOD. The change in optical density at 550 nm was quantified using the kinetics of cytochrome c reduction. To eliminate superoxide-independent cytochrome c reduction, the absorbance value with PEG-SOD was subtracted from the absorbance value with cytochrome c alone for each cell line system .

    Journal: PLoS ONE

    Article Title: NADPH oxidase 1 is highly expressed in human large and small bowel cancers

    doi: 10.1371/journal.pone.0233208

    Figure Lengend Snippet: Functional expression of NOX1 isoforms in human colon cancer cell lines. (A) RT-PCR and subsequent DNA gel analyses were performed to detect NOX1-L and NOX1-S / NOX1-Lv in a subset of 8 colon cancer cell lines plus HEK293-NOX1. (B) The subset of cell lines was evaluated for PMA-stimulated superoxide production by luminescence assay. (C-G) Basal and PMA-stimulated superoxide production were also measured in (C) HEK293-vector control, (D) HEK293-NOX1, (E) LS513, (F) HT-29, and (G) RKO cells using the luminol assay. Superoxide dismutase-polyethylene glycol (PEG-SOD, 200 U/ml) was used to confirm superoxide production, with measurements taken every 2 min for up to 120 min. (H) Mean rate of superoxide production at 60 min in nmol/h/10 6 cells. 2X10 6 cells per well were suspended in 200 μl of HBSS-HEPES containing 100 μM acetylated cytochrome c with or without 200 nM PMA and/or 200 U/ml PEG-SOD. The change in optical density at 550 nm was quantified using the kinetics of cytochrome c reduction. To eliminate superoxide-independent cytochrome c reduction, the absorbance value with PEG-SOD was subtracted from the absorbance value with cytochrome c alone for each cell line system .

    Article Snippet: The change in optical density at 550 nm was quantified at 60 min using the known kinetics of cytochrome c reduction [ , ].

    Techniques: Functional Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Luminescence Assay, Plasmid Preparation

    Cortical 45-kDa Gαs is contained in low-density membrane and is reduced after treatment of oocytes with progesterone or MeβCD

    Journal: Developmental biology

    Article Title: Activation of the progesterone signaling pathway by methyl-?-cyclodextrin or steroid in Xenopus laevis oocytes involves release of 45-kDa G?s

    doi: 10.1016/j.ydbio.2008.07.031

    Figure Lengend Snippet: Cortical 45-kDa Gαs is contained in low-density membrane and is reduced after treatment of oocytes with progesterone or MeβCD

    Article Snippet: Treatment of oocytes with progesterone or MeβCD reduced the apparent amounts of 45-kDa Gαs detected in oocyte cortices with the Calbiochem anti-Gαs antibody ( ).

    Techniques:

    Progesterone- and MeβCD-stimulated increases in oocyte 39-kDa Mos are inhibited by cycloheximide

    Journal: Developmental biology

    Article Title: Activation of the progesterone signaling pathway by methyl-?-cyclodextrin or steroid in Xenopus laevis oocytes involves release of 45-kDa G?s

    doi: 10.1016/j.ydbio.2008.07.031

    Figure Lengend Snippet: Progesterone- and MeβCD-stimulated increases in oocyte 39-kDa Mos are inhibited by cycloheximide

    Article Snippet: Treatment of oocytes with progesterone or MeβCD reduced the apparent amounts of 45-kDa Gαs detected in oocyte cortices with the Calbiochem anti-Gαs antibody ( ).

    Techniques:

    The steroid synthesis inhibitor, aminoglutethimide, does not block oocyte maturation stimulated MeβCD

    Journal: Developmental biology

    Article Title: Activation of the progesterone signaling pathway by methyl-?-cyclodextrin or steroid in Xenopus laevis oocytes involves release of 45-kDa G?s

    doi: 10.1016/j.ydbio.2008.07.031

    Figure Lengend Snippet: The steroid synthesis inhibitor, aminoglutethimide, does not block oocyte maturation stimulated MeβCD

    Article Snippet: Treatment of oocytes with progesterone or MeβCD reduced the apparent amounts of 45-kDa Gαs detected in oocyte cortices with the Calbiochem anti-Gαs antibody ( ).

    Techniques: Blocking Assay

    Follicle cells are not required for phosphorylation of oocyte MAPK or GVBD induced by MeβCD

    Journal: Developmental biology

    Article Title: Activation of the progesterone signaling pathway by methyl-?-cyclodextrin or steroid in Xenopus laevis oocytes involves release of 45-kDa G?s

    doi: 10.1016/j.ydbio.2008.07.031

    Figure Lengend Snippet: Follicle cells are not required for phosphorylation of oocyte MAPK or GVBD induced by MeβCD

    Article Snippet: Treatment of oocytes with progesterone or MeβCD reduced the apparent amounts of 45-kDa Gαs detected in oocyte cortices with the Calbiochem anti-Gαs antibody ( ).

    Techniques:

    Cycloheximide blocks MAPK phosphorylation and GVBD in response to MeβCD

    Journal: Developmental biology

    Article Title: Activation of the progesterone signaling pathway by methyl-?-cyclodextrin or steroid in Xenopus laevis oocytes involves release of 45-kDa G?s

    doi: 10.1016/j.ydbio.2008.07.031

    Figure Lengend Snippet: Cycloheximide blocks MAPK phosphorylation and GVBD in response to MeβCD

    Article Snippet: Treatment of oocytes with progesterone or MeβCD reduced the apparent amounts of 45-kDa Gαs detected in oocyte cortices with the Calbiochem anti-Gαs antibody ( ).

    Techniques:

    Recovery of dysregulated protein signature in NPC1 fibroblasts upon MβCD treatment. (A) Heat map showing the identified and quantified proteins that are differentially expressed in NPC1 fibroblast cells, comparing with WT, and reversed by MβCD-1, MβCD-2, or MβCD-3 treatments. The color key indicates the relative abundance of proteins (0–1.0) across five samples. Relative protein levels of NPC2 (B) , USE1 (C) , VAMP7 (D) , GABARAP (E) , NSDHL (F) , and DHCR24 (G) . CD, cyclodextrin; ppm, part per million.

    Journal: Assay and Drug Development Technologies

    Article Title: Analytical Characterization of Methyl-β-Cyclodextrin for Pharmacological Activity to Reduce Lysosomal Cholesterol Accumulation in Niemann-Pick Disease Type C1 Cells

    doi: 10.1089/adt.2017.774

    Figure Lengend Snippet: Recovery of dysregulated protein signature in NPC1 fibroblasts upon MβCD treatment. (A) Heat map showing the identified and quantified proteins that are differentially expressed in NPC1 fibroblast cells, comparing with WT, and reversed by MβCD-1, MβCD-2, or MβCD-3 treatments. The color key indicates the relative abundance of proteins (0–1.0) across five samples. Relative protein levels of NPC2 (B) , USE1 (C) , VAMP7 (D) , GABARAP (E) , NSDHL (F) , and DHCR24 (G) . CD, cyclodextrin; ppm, part per million.

    Article Snippet: Initially, 0.4 mg of MβCD powder was dissolved in 3 mL of deionized Millipore (Sigma-Aldrich, St. Louis, MO) water as the stock solution (100 μM).

    Techniques:

    Effects of different sources of MβCDs on reducing lysosome size in NPC1 fibroblasts. NPC1 patient skin fibroblasts (GM03123) and WT control (GM05659) were untreated or treated with MβCD (0.4–300 μM) for 4 days, after which LysoTracker ® staining was performed. (A) Images of LysoTracker staining on NPC1 fibroblasts. Treatment with 300 and 11 μM of MβCD-3 significantly reduced the cholesterol accumulation in NPC1 patient skin fibroblasts, while there were no significant effects observed from the other two batches of MβCDs (MβCD-1 and MβCD-2). LysoTracker red stains cellular acidic compartments to visualize enlarged lysosomes, and Hoechst ( blue ) stains nuclei. (B) Treatment with MβCD-3 (300 μM) significantly reduced the lysosome size in the NPC1 patient fibroblast compared with the other two batches of MβCDs (MβCD-1 and MβCD-2). (C) Dose–response curve of different sources of MβCDs on NPC1 patient fibroblasts. MβCD-3 showed concentration-dependent impact on lysosome size of NPC1 fibroblasts, while there were no significant effects observed from the other two batches of MβCDs.

    Journal: Assay and Drug Development Technologies

    Article Title: Analytical Characterization of Methyl-β-Cyclodextrin for Pharmacological Activity to Reduce Lysosomal Cholesterol Accumulation in Niemann-Pick Disease Type C1 Cells

    doi: 10.1089/adt.2017.774

    Figure Lengend Snippet: Effects of different sources of MβCDs on reducing lysosome size in NPC1 fibroblasts. NPC1 patient skin fibroblasts (GM03123) and WT control (GM05659) were untreated or treated with MβCD (0.4–300 μM) for 4 days, after which LysoTracker ® staining was performed. (A) Images of LysoTracker staining on NPC1 fibroblasts. Treatment with 300 and 11 μM of MβCD-3 significantly reduced the cholesterol accumulation in NPC1 patient skin fibroblasts, while there were no significant effects observed from the other two batches of MβCDs (MβCD-1 and MβCD-2). LysoTracker red stains cellular acidic compartments to visualize enlarged lysosomes, and Hoechst ( blue ) stains nuclei. (B) Treatment with MβCD-3 (300 μM) significantly reduced the lysosome size in the NPC1 patient fibroblast compared with the other two batches of MβCDs (MβCD-1 and MβCD-2). (C) Dose–response curve of different sources of MβCDs on NPC1 patient fibroblasts. MβCD-3 showed concentration-dependent impact on lysosome size of NPC1 fibroblasts, while there were no significant effects observed from the other two batches of MβCDs.

    Article Snippet: Initially, 0.4 mg of MβCD powder was dissolved in 3 mL of deionized Millipore (Sigma-Aldrich, St. Louis, MO) water as the stock solution (100 μM).

    Techniques: Staining, Concentration Assay

    Mass spectrometry on MβCD. Mass spectra of MβCD-1, -2, and -3 show cluster signals of sodium adduct ions of 6–12 different mixtures of methyl-substituted β-cyclodextrin molecules. Methylation number can be easily determined by an inclement of mass unit (m/z) 14 as shown. The mass spectrometry peak heights are proportional to molecular distributions (or amounts) of various methyl-substituted β-cyclodextrins (summarized in Table 1 ). Abundance of lower number of methyl substitution of MβCD is shown in (A) (MβCD-1), while middle substitution is in (B) (MβCD-2) and higher substitution is in (C) (MβCD-3). 10-Me, 10 methylation to β-cyclodextrin molecule.

    Journal: Assay and Drug Development Technologies

    Article Title: Analytical Characterization of Methyl-β-Cyclodextrin for Pharmacological Activity to Reduce Lysosomal Cholesterol Accumulation in Niemann-Pick Disease Type C1 Cells

    doi: 10.1089/adt.2017.774

    Figure Lengend Snippet: Mass spectrometry on MβCD. Mass spectra of MβCD-1, -2, and -3 show cluster signals of sodium adduct ions of 6–12 different mixtures of methyl-substituted β-cyclodextrin molecules. Methylation number can be easily determined by an inclement of mass unit (m/z) 14 as shown. The mass spectrometry peak heights are proportional to molecular distributions (or amounts) of various methyl-substituted β-cyclodextrins (summarized in Table 1 ). Abundance of lower number of methyl substitution of MβCD is shown in (A) (MβCD-1), while middle substitution is in (B) (MβCD-2) and higher substitution is in (C) (MβCD-3). 10-Me, 10 methylation to β-cyclodextrin molecule.

    Article Snippet: Initially, 0.4 mg of MβCD powder was dissolved in 3 mL of deionized Millipore (Sigma-Aldrich, St. Louis, MO) water as the stock solution (100 μM).

    Techniques: Mass Spectrometry, Methylation

    Chemical structure of β-cyclodextrin. (A) Chemical representation of methyl-β-cyclodextrin (MβCD), which comprises seven glucopyranose units. (B) Three-dimensional representation of the toroid structure of cyclodextrin consisting of a hydrophilic exterior and hydrophobic interior.

    Journal: Assay and Drug Development Technologies

    Article Title: Analytical Characterization of Methyl-β-Cyclodextrin for Pharmacological Activity to Reduce Lysosomal Cholesterol Accumulation in Niemann-Pick Disease Type C1 Cells

    doi: 10.1089/adt.2017.774

    Figure Lengend Snippet: Chemical structure of β-cyclodextrin. (A) Chemical representation of methyl-β-cyclodextrin (MβCD), which comprises seven glucopyranose units. (B) Three-dimensional representation of the toroid structure of cyclodextrin consisting of a hydrophilic exterior and hydrophobic interior.

    Article Snippet: Initially, 0.4 mg of MβCD powder was dissolved in 3 mL of deionized Millipore (Sigma-Aldrich, St. Louis, MO) water as the stock solution (100 μM).

    Techniques:

    Effects of different sources of MβCDs on reducing cholesterol accumulation in NPC1 fibroblasts. NPC1 patient skin fibroblasts (GM03123) and WT control (GM05659) were untreated or treated with MβCD (0.4–300 μM) for 4 days; filipin staining was then performed. (A) Images of filipin staining on NPC1 fibroblasts. Treatment with 300, 11 μM of MβCD-3 significantly reduced cholesterol accumulation in NPC1 patient skin fibroblasts, while the other two batches of MβCDs (MβCD-1 and MβCD-2) showed much weaker effects on cholesterol accumulation in NPC1 patient fibroblasts. Filipin ( green ) stains the intracellular cholesterol-laden domains, and EthD-1 ( red ) stains nuclei. (B) Treatment with MβCD-3 (300 μM) significantly reduced cholesterol accumulation in the NPC1 patient fibroblast compared with the other two batches of MβCDs (MβCD-1 and MβCD-2). (C) Dose–response curve of different sources of MβCDs on cholesterol accumulation in NPC1 patient fibroblasts. EthD-1, ethidium homodimer; NPC1, Niemann-Pick disease type C1; WT, wild-type.

    Journal: Assay and Drug Development Technologies

    Article Title: Analytical Characterization of Methyl-β-Cyclodextrin for Pharmacological Activity to Reduce Lysosomal Cholesterol Accumulation in Niemann-Pick Disease Type C1 Cells

    doi: 10.1089/adt.2017.774

    Figure Lengend Snippet: Effects of different sources of MβCDs on reducing cholesterol accumulation in NPC1 fibroblasts. NPC1 patient skin fibroblasts (GM03123) and WT control (GM05659) were untreated or treated with MβCD (0.4–300 μM) for 4 days; filipin staining was then performed. (A) Images of filipin staining on NPC1 fibroblasts. Treatment with 300, 11 μM of MβCD-3 significantly reduced cholesterol accumulation in NPC1 patient skin fibroblasts, while the other two batches of MβCDs (MβCD-1 and MβCD-2) showed much weaker effects on cholesterol accumulation in NPC1 patient fibroblasts. Filipin ( green ) stains the intracellular cholesterol-laden domains, and EthD-1 ( red ) stains nuclei. (B) Treatment with MβCD-3 (300 μM) significantly reduced cholesterol accumulation in the NPC1 patient fibroblast compared with the other two batches of MβCDs (MβCD-1 and MβCD-2). (C) Dose–response curve of different sources of MβCDs on cholesterol accumulation in NPC1 patient fibroblasts. EthD-1, ethidium homodimer; NPC1, Niemann-Pick disease type C1; WT, wild-type.

    Article Snippet: Initially, 0.4 mg of MβCD powder was dissolved in 3 mL of deionized Millipore (Sigma-Aldrich, St. Louis, MO) water as the stock solution (100 μM).

    Techniques: Staining, Ethidium Homodimer Assay

    Effect of different sources of MβCDs on cholesterol accumulation in NPC1 fibroblasts. NPC1 patient skin fibroblasts (GM03123) were treated with MβCD (0.4–300 μM) for 4 days, followed by an Amplex ® Red cholesterol assay. (A) Dose–response curve of different sources of MβCDs on NPC1 patient fibroblasts on cholesterol accumulation in NPC1 fibroblasts. MβCD-3 showed concentration-dependent manner on cholesterol accumulation in NPC1 fibroblasts GM03123, while there were much weaker effects observed with the other two batches of MβCDs. The maximum inhibitory effect of MβCD-3 is about 46.0% compared with MβCD-1, 15.2%, and MβCD-2, 19.5%. (B) Treatment with 300 μM of MβCD-3 significantly reduced cholesterol accumulation in NPC1 fibroblasts compared with the other two sources of MβCDs. (C) Cytotoxicity (ATPlite assay) of different sources of MβCDs on NPC1 fibroblasts. NPC1 patient skin fibroblasts (GM03123) were untreated or treated with MβCD (0.4–300 μM) for 4 days and ATPlite assay was performed to evaluate the cytotoxic effects of different sources of MβCDs on the fibroblasts. There were no significant cytotoxic effects observed within the test range of 0.4–300 μM of MβCD and the cell viability level was generally above 90% after 4 days of treatment.

    Journal: Assay and Drug Development Technologies

    Article Title: Analytical Characterization of Methyl-β-Cyclodextrin for Pharmacological Activity to Reduce Lysosomal Cholesterol Accumulation in Niemann-Pick Disease Type C1 Cells

    doi: 10.1089/adt.2017.774

    Figure Lengend Snippet: Effect of different sources of MβCDs on cholesterol accumulation in NPC1 fibroblasts. NPC1 patient skin fibroblasts (GM03123) were treated with MβCD (0.4–300 μM) for 4 days, followed by an Amplex ® Red cholesterol assay. (A) Dose–response curve of different sources of MβCDs on NPC1 patient fibroblasts on cholesterol accumulation in NPC1 fibroblasts. MβCD-3 showed concentration-dependent manner on cholesterol accumulation in NPC1 fibroblasts GM03123, while there were much weaker effects observed with the other two batches of MβCDs. The maximum inhibitory effect of MβCD-3 is about 46.0% compared with MβCD-1, 15.2%, and MβCD-2, 19.5%. (B) Treatment with 300 μM of MβCD-3 significantly reduced cholesterol accumulation in NPC1 fibroblasts compared with the other two sources of MβCDs. (C) Cytotoxicity (ATPlite assay) of different sources of MβCDs on NPC1 fibroblasts. NPC1 patient skin fibroblasts (GM03123) were untreated or treated with MβCD (0.4–300 μM) for 4 days and ATPlite assay was performed to evaluate the cytotoxic effects of different sources of MβCDs on the fibroblasts. There were no significant cytotoxic effects observed within the test range of 0.4–300 μM of MβCD and the cell viability level was generally above 90% after 4 days of treatment.

    Article Snippet: Initially, 0.4 mg of MβCD powder was dissolved in 3 mL of deionized Millipore (Sigma-Aldrich, St. Louis, MO) water as the stock solution (100 μM).

    Techniques: Amplex Red Cholesterol Assay, Concentration Assay