Structured Review

Millipore dmem
Fusion from without of <t>BVDV</t> in the presence or absence of DTT. MDBK cells were inoculated with BVDV strain NADL and briefly shifted to 37°C at the indicated pH in the presence or absence of 10 mM DTT; virus uptake via endocytosis was blocked by replacing buffer with <t>DMEM</t> containing bafilomycin A1. Higher concentrations of DTT could not be used due to high cell toxicity. The numbers of infectious centers were determined 12 to 16 h p.i. The columns represent mean values of triplicate experiments; bars indicate maximum and minimum values.
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Images

1) Product Images from "Acid-Resistant Bovine Pestivirus Requires Activation for pH-Triggered Fusion during Entry"

Article Title: Acid-Resistant Bovine Pestivirus Requires Activation for pH-Triggered Fusion during Entry

Journal: Journal of Virology

doi: 10.1128/JVI.79.7.4191-4200.2005

Fusion from without of BVDV in the presence or absence of DTT. MDBK cells were inoculated with BVDV strain NADL and briefly shifted to 37°C at the indicated pH in the presence or absence of 10 mM DTT; virus uptake via endocytosis was blocked by replacing buffer with DMEM containing bafilomycin A1. Higher concentrations of DTT could not be used due to high cell toxicity. The numbers of infectious centers were determined 12 to 16 h p.i. The columns represent mean values of triplicate experiments; bars indicate maximum and minimum values.
Figure Legend Snippet: Fusion from without of BVDV in the presence or absence of DTT. MDBK cells were inoculated with BVDV strain NADL and briefly shifted to 37°C at the indicated pH in the presence or absence of 10 mM DTT; virus uptake via endocytosis was blocked by replacing buffer with DMEM containing bafilomycin A1. Higher concentrations of DTT could not be used due to high cell toxicity. The numbers of infectious centers were determined 12 to 16 h p.i. The columns represent mean values of triplicate experiments; bars indicate maximum and minimum values.

Techniques Used:

Fusion from without of BVDV and SinV. MDBK cells were inoculated with BVDV strain NADL or SinV, respectively, for 1 h at 4°C. Medium was replaced by prewarmed buffers of the indicated pH, followed by incubation for 2 min at 37°C. Viral uptake via endocytosis was blocked by replacing buffer with DMEM containing bafilomycin A1. Since fusion from without is cell type specific, SinV was used as control. The columns represent mean values of triplicate experiments; bars indicate maximum and minimum values.
Figure Legend Snippet: Fusion from without of BVDV and SinV. MDBK cells were inoculated with BVDV strain NADL or SinV, respectively, for 1 h at 4°C. Medium was replaced by prewarmed buffers of the indicated pH, followed by incubation for 2 min at 37°C. Viral uptake via endocytosis was blocked by replacing buffer with DMEM containing bafilomycin A1. Since fusion from without is cell type specific, SinV was used as control. The columns represent mean values of triplicate experiments; bars indicate maximum and minimum values.

Techniques Used: Incubation

2) Product Images from "Single-molecule analysis of CD9 dynamics and partitioning reveals multiple modes of interaction in the tetraspanin web"

Article Title: Single-molecule analysis of CD9 dynamics and partitioning reveals multiple modes of interaction in the tetraspanin web

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200803010

Distribution of the ADC and diffusion modes of CD9 and CD9 plm and their partitioning in tetraspanin-enriched compartments. (left) ADC distribution of CD9 in control cells (CD9), cells treated with MβCD (CD9 MβCD), cells treated with MβCD loaded with Chl (CD9 MβCD–Chl), or cells transfected with nonpalmitoylated CD9 (CD9 plm ). 50% of the membrane Chl was removed by MβCD treatment, and MβCD–Chl treatment increased the Chl content to 130% as compared with control cells. All of the palmitoylation sites have been mutated in CD9 plm cells. (right) Histograms (open boxes) representing the percentage of each diffusion mode of the molecules as compared with the total number of trajectories (B, Brownian; C, confined; M, mixed). The gray part corresponds to the proportion of trajectories associated with TEAs (identified with the ensemble membrane labeling) for each diffusion mode.
Figure Legend Snippet: Distribution of the ADC and diffusion modes of CD9 and CD9 plm and their partitioning in tetraspanin-enriched compartments. (left) ADC distribution of CD9 in control cells (CD9), cells treated with MβCD (CD9 MβCD), cells treated with MβCD loaded with Chl (CD9 MβCD–Chl), or cells transfected with nonpalmitoylated CD9 (CD9 plm ). 50% of the membrane Chl was removed by MβCD treatment, and MβCD–Chl treatment increased the Chl content to 130% as compared with control cells. All of the palmitoylation sites have been mutated in CD9 plm cells. (right) Histograms (open boxes) representing the percentage of each diffusion mode of the molecules as compared with the total number of trajectories (B, Brownian; C, confined; M, mixed). The gray part corresponds to the proportion of trajectories associated with TEAs (identified with the ensemble membrane labeling) for each diffusion mode.

Techniques Used: Diffusion-based Assay, Transfection, Labeling

Influence of MβCD on membrane dynamics. (A) Distribution of the ADC of CD9, CD55, and CD46 treated or not treated with MβCD (∼50% of the membrane Chl was removed). CD55 is a raft marker, and CD46 is excluded from rafts and TEAs. Mean values of ADC of all the molecules are available in Table I . (B) Comparison of trajectories (thin white lines) in living PC3 cells before (left) or after (right) MβCD treatment. Bars, 7.5 μm.
Figure Legend Snippet: Influence of MβCD on membrane dynamics. (A) Distribution of the ADC of CD9, CD55, and CD46 treated or not treated with MβCD (∼50% of the membrane Chl was removed). CD55 is a raft marker, and CD46 is excluded from rafts and TEAs. Mean values of ADC of all the molecules are available in Table I . (B) Comparison of trajectories (thin white lines) in living PC3 cells before (left) or after (right) MβCD treatment. Bars, 7.5 μm.

Techniques Used: Marker

3) Product Images from "Identification of Caveolar Resident Proteins in Ventricular Myocytes Using a Quantitative Proteomic Approach: Dynamic Changes in Caveolar Composition Following Adrenoceptor Activation *"

Article Title: Identification of Caveolar Resident Proteins in Ventricular Myocytes Using a Quantitative Proteomic Approach: Dynamic Changes in Caveolar Composition Following Adrenoceptor Activation *

Journal: Molecular & Cellular Proteomics : MCP

doi: 10.1074/mcp.M114.038570

iTraq analysis of control and MβCD treated BCEMs. A , Distribution of iTraq ratios from two independent experiments before (left) and after (right) normalization. B , After normalization, iTRAQ ratios show good reproducibility between experiments. Proteins annotated as caveolar residents in Uniprot are highlighted in red. The caveolar proteome was defined as the boxed area. C , Candidate caveolar proteins were verified by immunoblotting fraction 4 purified from control and MβCD treated cells. SM = gradient starting material.
Figure Legend Snippet: iTraq analysis of control and MβCD treated BCEMs. A , Distribution of iTraq ratios from two independent experiments before (left) and after (right) normalization. B , After normalization, iTRAQ ratios show good reproducibility between experiments. Proteins annotated as caveolar residents in Uniprot are highlighted in red. The caveolar proteome was defined as the boxed area. C , Candidate caveolar proteins were verified by immunoblotting fraction 4 purified from control and MβCD treated cells. SM = gradient starting material.

Techniques Used: Purification

Depletion of caveolin 3 and cholesterol, but not bulk sarcolemmal or mitochondrial proteins from buoyant membranes following MβCD treatment of adult rat ventricular myocytes ( n = 5 per group for all measurements, mean ± S. E. shown) . A , Buoyant membranes were prepared using a discontinuous sucrose gradient. B , Caveolae were isolated from control (left) and MβCD-treated (right) adult rat ventricular myocytes. Fractions were immunoblotted for the proteins indicated. C , Quantitative assessment of the depletion of caveolin 3 from gradient fractions 4 and 5 following MβCD treatment. D , Quantitative assessment of the distribution of cholesterol in all gradient fractions (control left, MβCD right). E , Depletion of protein from gradient fractions 4 and 5 following MβCD treatment. F , Distribution of protein for all gradient fractions (control left, MβCD right).
Figure Legend Snippet: Depletion of caveolin 3 and cholesterol, but not bulk sarcolemmal or mitochondrial proteins from buoyant membranes following MβCD treatment of adult rat ventricular myocytes ( n = 5 per group for all measurements, mean ± S. E. shown) . A , Buoyant membranes were prepared using a discontinuous sucrose gradient. B , Caveolae were isolated from control (left) and MβCD-treated (right) adult rat ventricular myocytes. Fractions were immunoblotted for the proteins indicated. C , Quantitative assessment of the depletion of caveolin 3 from gradient fractions 4 and 5 following MβCD treatment. D , Quantitative assessment of the distribution of cholesterol in all gradient fractions (control left, MβCD right). E , Depletion of protein from gradient fractions 4 and 5 following MβCD treatment. F , Distribution of protein for all gradient fractions (control left, MβCD right).

Techniques Used: Isolation

4) Product Images from "Neuropilin 1 is an entry factor that promotes EBV infection of nasopharyngeal epithelial cells"

Article Title: Neuropilin 1 is an entry factor that promotes EBV infection of nasopharyngeal epithelial cells

Journal: Nature Communications

doi: 10.1038/ncomms7240

EBV enters epithelial cells via lipid raft-dependent endocytosis and macropinocytosis. ( a , b ) EBV co-localized with EGFP-SNX5, but not EGFP-CLCa. HNE1 cells transiently transfected with expression vectors for EGFP-SNX5 ( a ) or EGFP-CLCa ( b ) for 24 h were infected with Alexa fluor 594 labelled EBV (EBV-Alex 594) for 60 min at 37 °C. ( c , d ) EGFP-SNX5, but not EGFP-CLCa co-localized with NRP1. HNE1 cells transiently co-transfected with the expression vectors for NRP1-mCherry and EGFP-SNX5 ( c ) or EGFP-CLCa ( d ) for 24 h were infected with cell-free EBV. For ( a – d ), expressions of EGFP-SNX5 and EGFP-CLCa were visualized as green, and EBV-Alexa fluor 594 and expression of NRP1-mCherry were imaged as red. Nuclei were stained with DAPI (blue). ( e – g ) EBV infection was dose-dependently suppressed by EIPA ( e ) and MβCD ( f ), but not CPZ ( g ). EGF-treated HNE1 cells and NPEC1-Bmi1 cells were pre-incubated with the indicated doses of EIPA, MβCD and CPZ for 30 min, followed by EBV infection for 2 h. The cells were then washed with Hanks solution twice and cultured for 48 h until FACS analysis; n =3. Graphs show mean±s.e.m. *** P
Figure Legend Snippet: EBV enters epithelial cells via lipid raft-dependent endocytosis and macropinocytosis. ( a , b ) EBV co-localized with EGFP-SNX5, but not EGFP-CLCa. HNE1 cells transiently transfected with expression vectors for EGFP-SNX5 ( a ) or EGFP-CLCa ( b ) for 24 h were infected with Alexa fluor 594 labelled EBV (EBV-Alex 594) for 60 min at 37 °C. ( c , d ) EGFP-SNX5, but not EGFP-CLCa co-localized with NRP1. HNE1 cells transiently co-transfected with the expression vectors for NRP1-mCherry and EGFP-SNX5 ( c ) or EGFP-CLCa ( d ) for 24 h were infected with cell-free EBV. For ( a – d ), expressions of EGFP-SNX5 and EGFP-CLCa were visualized as green, and EBV-Alexa fluor 594 and expression of NRP1-mCherry were imaged as red. Nuclei were stained with DAPI (blue). ( e – g ) EBV infection was dose-dependently suppressed by EIPA ( e ) and MβCD ( f ), but not CPZ ( g ). EGF-treated HNE1 cells and NPEC1-Bmi1 cells were pre-incubated with the indicated doses of EIPA, MβCD and CPZ for 30 min, followed by EBV infection for 2 h. The cells were then washed with Hanks solution twice and cultured for 48 h until FACS analysis; n =3. Graphs show mean±s.e.m. *** P

Techniques Used: Transfection, Expressing, Infection, Staining, Incubation, Cell Culture, FACS

5) 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

6) Product Images from "Endocytosis of Chikungunya Virus into Mammalian Cells: Role of Clathrin and Early Endosomal Compartments"

Article Title: Endocytosis of Chikungunya Virus into Mammalian Cells: Role of Clathrin and Early Endosomal Compartments

Journal: PLoS ONE

doi: 10.1371/journal.pone.0011479

Functional Eps15 but not clathrin heavy chain expression is required for infection by CHIKV. HEK293T cells were transfected with plasmids encoding either a GFP-labelled wild type Eps15 (WT) or the GFP-tagged dominant negative Eps15Δ95/295 mutant (DN) and maintained for 36 h in culture. (A) Cells were incubated with Alexa595-labelled transferrin for 30 min at 37°C and uptake by transfected cells was monitored using immunofluorescence microscopy. (B) Transfected cells were challenged with CHIKV for 4 h at 37°C (37997-CHIKV strain used at a m.o.i. of 5) and the presence of intracellular viral antigens was detected using a mAbs C42 reacting with CHIKV and Texas-red-conjugated secondary antibodies. (C) Tansgene-expressing cells positive for intracellular CHIKV antigens were quantified from panel (B) by flow cytometry. Values are the mean of 4 separate experiments performed in triplicate ± SD. (D) Cells transfected with siRNA targeting the clathrin heavy chain (CHC) or with control siRNA (Ctrl.) were challenged with 37997-CHIKV. Levels of infection were monitored by quantification of GFP-positive cells using flow cytometry (upper panel). Levels of CHC expression in transfected cells were analyzed by immunoblot analysis. Actin expression was used to monitor proteins level in each sample (lower panel).
Figure Legend Snippet: Functional Eps15 but not clathrin heavy chain expression is required for infection by CHIKV. HEK293T cells were transfected with plasmids encoding either a GFP-labelled wild type Eps15 (WT) or the GFP-tagged dominant negative Eps15Δ95/295 mutant (DN) and maintained for 36 h in culture. (A) Cells were incubated with Alexa595-labelled transferrin for 30 min at 37°C and uptake by transfected cells was monitored using immunofluorescence microscopy. (B) Transfected cells were challenged with CHIKV for 4 h at 37°C (37997-CHIKV strain used at a m.o.i. of 5) and the presence of intracellular viral antigens was detected using a mAbs C42 reacting with CHIKV and Texas-red-conjugated secondary antibodies. (C) Tansgene-expressing cells positive for intracellular CHIKV antigens were quantified from panel (B) by flow cytometry. Values are the mean of 4 separate experiments performed in triplicate ± SD. (D) Cells transfected with siRNA targeting the clathrin heavy chain (CHC) or with control siRNA (Ctrl.) were challenged with 37997-CHIKV. Levels of infection were monitored by quantification of GFP-positive cells using flow cytometry (upper panel). Levels of CHC expression in transfected cells were analyzed by immunoblot analysis. Actin expression was used to monitor proteins level in each sample (lower panel).

Techniques Used: Functional Assay, Expressing, Infection, Transfection, Dominant Negative Mutation, Mutagenesis, Incubation, Immunofluorescence, Microscopy, Flow Cytometry, Cytometry

Comparative analysis of entry pathways used by the 37997 African strain and the LR-OPY1 isolate to infect HEK293T cells. Cells were challenged with normalized amounts (m.o.i. of 5) of the 37997-GFP (black bars) or the LR-OPY1-GFP (white bars) strains of CHIKV after (A) transfection of cells with siRNA directed to clathrin heavy chain (CHC) or control siRNA (Ctrl), or after treatment with (B) lysomotropic agents (chloroquine (Chl), monensin (Mo), amonium chloride (NH 4 Cl) or bafilomycin A1 (Baf)) or (C) cytochalasine D (CytD) or (D) nocodazole (NoD) in conditions described in Material and Methods . For each experiment, a control performed by incubating the cells in the presence of medium supplemented with an equivalent amount of drug solvent (Med) is included. Twenty-four hours post-infection, CHIKV replication was monitored by flow cytometry analysis of GFP expression. Each value is the mean of 3 separate experiments performed in triplicate ± SD.
Figure Legend Snippet: Comparative analysis of entry pathways used by the 37997 African strain and the LR-OPY1 isolate to infect HEK293T cells. Cells were challenged with normalized amounts (m.o.i. of 5) of the 37997-GFP (black bars) or the LR-OPY1-GFP (white bars) strains of CHIKV after (A) transfection of cells with siRNA directed to clathrin heavy chain (CHC) or control siRNA (Ctrl), or after treatment with (B) lysomotropic agents (chloroquine (Chl), monensin (Mo), amonium chloride (NH 4 Cl) or bafilomycin A1 (Baf)) or (C) cytochalasine D (CytD) or (D) nocodazole (NoD) in conditions described in Material and Methods . For each experiment, a control performed by incubating the cells in the presence of medium supplemented with an equivalent amount of drug solvent (Med) is included. Twenty-four hours post-infection, CHIKV replication was monitored by flow cytometry analysis of GFP expression. Each value is the mean of 3 separate experiments performed in triplicate ± SD.

Techniques Used: Transfection, Infection, Flow Cytometry, Cytometry, Expressing

Role of Rab5 and Rab7 GTPases in CHIKV infection of HEK293T cells. HEK293T cells grown on coverslips were transfected with plasmids encoding either wild-type (WT) or dominant negative (DN) forms of GFP-tagged Rab5 (A), (B) and (D) or GFP-tagged Rab7 (C) and (E). Twenty-four hours post-transfection, cells were challenged with CHIKV (37997 strain used at a m.o.i. of 5). After another 4 hours in culture, the cells were permeabilized and viral antigens were labelled using anti-capsid C42 mAb and Alexa 594 secondary anti-Ig reagents. Antigen-expressing cells were revealed by confocal imaging (B) and (C), or quantified by flow cytometry (D) and (E). In control experiments, cells expressing GFP-fused WT or DN Rab5 were analyzed for Alexa Fluor 594-labelled transferrin uptake by confocal imaging (A). All experiments were repeated three times with similar outcomes. Images are representative of individual cells visualized and percentages of CHIKV positive cells determined by flow cytometry analysis are the mean of 4 separate experiments performed in triplicate ± SD.
Figure Legend Snippet: Role of Rab5 and Rab7 GTPases in CHIKV infection of HEK293T cells. HEK293T cells grown on coverslips were transfected with plasmids encoding either wild-type (WT) or dominant negative (DN) forms of GFP-tagged Rab5 (A), (B) and (D) or GFP-tagged Rab7 (C) and (E). Twenty-four hours post-transfection, cells were challenged with CHIKV (37997 strain used at a m.o.i. of 5). After another 4 hours in culture, the cells were permeabilized and viral antigens were labelled using anti-capsid C42 mAb and Alexa 594 secondary anti-Ig reagents. Antigen-expressing cells were revealed by confocal imaging (B) and (C), or quantified by flow cytometry (D) and (E). In control experiments, cells expressing GFP-fused WT or DN Rab5 were analyzed for Alexa Fluor 594-labelled transferrin uptake by confocal imaging (A). All experiments were repeated three times with similar outcomes. Images are representative of individual cells visualized and percentages of CHIKV positive cells determined by flow cytometry analysis are the mean of 4 separate experiments performed in triplicate ± SD.

Techniques Used: Infection, Transfection, Dominant Negative Mutation, Expressing, Imaging, Flow Cytometry, Cytometry

CHIKV infection of HEK293T cells is inhibited by lysomotropic agents. (A) Cells were incubated in medium supplemented with drug solvent (Med) or medium supplemented with appropriate concentrations of lysomotropic agents inhibiting endosomal acidification: chloroquine (Chl), monensine (Mo), amonium chloride (NH 4 Cl), bafilomycine A1 (Baf). The cells were challenged with the 37997-GFP CHIKV strain used at a m.o.i. of 5. The percentage of GFP-expressing cells was determined by flow cytometry analysis (black bars). For each experiment, the number of viable cells was determined after exclusion coloration with Trypan Blue (white bars). Values are expressed as a percentage of untreated controls. Each value is the mean of 3 separate experiments performed in triplicate ± SD. (B) Appropriate concentrations of lysomotropic drugs were added to CHIKV infected cells 4 hours after viral challenge (m.o.i. of 5). After an additional 16 h in culture, percentage of infected cells was determined by flow cytometry.
Figure Legend Snippet: CHIKV infection of HEK293T cells is inhibited by lysomotropic agents. (A) Cells were incubated in medium supplemented with drug solvent (Med) or medium supplemented with appropriate concentrations of lysomotropic agents inhibiting endosomal acidification: chloroquine (Chl), monensine (Mo), amonium chloride (NH 4 Cl), bafilomycine A1 (Baf). The cells were challenged with the 37997-GFP CHIKV strain used at a m.o.i. of 5. The percentage of GFP-expressing cells was determined by flow cytometry analysis (black bars). For each experiment, the number of viable cells was determined after exclusion coloration with Trypan Blue (white bars). Values are expressed as a percentage of untreated controls. Each value is the mean of 3 separate experiments performed in triplicate ± SD. (B) Appropriate concentrations of lysomotropic drugs were added to CHIKV infected cells 4 hours after viral challenge (m.o.i. of 5). After an additional 16 h in culture, percentage of infected cells was determined by flow cytometry.

Techniques Used: Infection, Incubation, Expressing, Flow Cytometry, Cytometry

Effect of actin-disrupting drug and microtubule depolymerizating agent on CHIKV infection. HEK293T cells were treated with increasing concentrations of (A) cytochalasin D (Cyt D) or (B) nocodazole (NoD) before exposure to the 37997-GFP strain of CHIKV (m.o.i. of 5) (black bars). Sixteen hours post-infection, the percentage of infected cells was determined by quantification of GFP-expressing cells by flow cytometry analysis. For each point, cell viability was determined by Trypan blue exclusion and expressed as a percentage of untreated controls (white bars). Values are the mean of 3 separate experiments performed in duplicate + SD. Organization of cytoskeleton in cells cultured in medium alone (Med) or supplemented with cytochalsin D (C) or nocodazole (D) was analyzed by immunofluorescence after labelling with anti-actin (C) or anti-tubulin mAbs (D). Nuclei are stained with DAPI. (E) Flow cytometry analysis of CHIKV infected cells after treatment with cytochalasine D or nocodazole and viral challenge performed in neutral (black bars) or acid (white bars) medium. Each value is the mean of 3 separate experiments performed in duplicate ± SD.
Figure Legend Snippet: Effect of actin-disrupting drug and microtubule depolymerizating agent on CHIKV infection. HEK293T cells were treated with increasing concentrations of (A) cytochalasin D (Cyt D) or (B) nocodazole (NoD) before exposure to the 37997-GFP strain of CHIKV (m.o.i. of 5) (black bars). Sixteen hours post-infection, the percentage of infected cells was determined by quantification of GFP-expressing cells by flow cytometry analysis. For each point, cell viability was determined by Trypan blue exclusion and expressed as a percentage of untreated controls (white bars). Values are the mean of 3 separate experiments performed in duplicate + SD. Organization of cytoskeleton in cells cultured in medium alone (Med) or supplemented with cytochalsin D (C) or nocodazole (D) was analyzed by immunofluorescence after labelling with anti-actin (C) or anti-tubulin mAbs (D). Nuclei are stained with DAPI. (E) Flow cytometry analysis of CHIKV infected cells after treatment with cytochalasine D or nocodazole and viral challenge performed in neutral (black bars) or acid (white bars) medium. Each value is the mean of 3 separate experiments performed in duplicate ± SD.

Techniques Used: Infection, Expressing, Flow Cytometry, Cytometry, Cell Culture, Immunofluorescence, Staining

Methyl β-cyclodextrin treatment decreases infection of mammalian cells by CHIKV. HEK293T cells were incubated in medium alone or medium supplemented with increasing concentrations of methyl β-cyclodextrin (mCD) and challenged with the 37997-GFP CHIKV strain (m.o.i. of 5). The percentage of GFP expressing cells was determined by flow cytometry analysis (black histograms). For each experiment, the number of viable cells was determined after exclusion coloration with Trypan Blue (white histograms). Values are expressed as a percentage of untreated conditions. Each value is the mean of 3 separate experiments performed in triplicate ± SD.
Figure Legend Snippet: Methyl β-cyclodextrin treatment decreases infection of mammalian cells by CHIKV. HEK293T cells were incubated in medium alone or medium supplemented with increasing concentrations of methyl β-cyclodextrin (mCD) and challenged with the 37997-GFP CHIKV strain (m.o.i. of 5). The percentage of GFP expressing cells was determined by flow cytometry analysis (black histograms). For each experiment, the number of viable cells was determined after exclusion coloration with Trypan Blue (white histograms). Values are expressed as a percentage of untreated conditions. Each value is the mean of 3 separate experiments performed in triplicate ± SD.

Techniques Used: Infection, Incubation, Expressing, Flow Cytometry, Cytometry

7) Product Images from "Akt Inhibition Promotes ABCA1-Mediated Cholesterol Efflux to ApoA-I through Suppressing mTORC1"

Article Title: Akt Inhibition Promotes ABCA1-Mediated Cholesterol Efflux to ApoA-I through Suppressing mTORC1

Journal: PLoS ONE

doi: 10.1371/journal.pone.0113789

Akt inhibition by LY294002 or Akt1/2 also enhances cholesterol efflux to apoA-I from ABCA1-expressing BHK cells. A ) BHK cells were induced as in Fig. 1 and then incubated with LY294002 (200 µM) for 2 h. Cells were then lysed and analysed for ABCA1, phosphorylated Akt (p-Akt) and total Akt by Western-blotting. Hsp70 was also blotted as loading control. B C ) BHK cells were labeled with [ 3 H] cholesterol and induced with 10 nM mifepristone overnight. Cholesterol efflux was measured after 2 h incubation with BSA (1 mg/ml) or BSA plus apoA-I (5 µg/ml). Some of cells were also incubated with indicated doses of LY294002 ( B ) or Akt1/2 ( C ), in addition to apoA-I, during 2 h efflux period. Results are presented as the average of triplicate wells with standard deviation and representative of more than three independent experiments. *** P
Figure Legend Snippet: Akt inhibition by LY294002 or Akt1/2 also enhances cholesterol efflux to apoA-I from ABCA1-expressing BHK cells. A ) BHK cells were induced as in Fig. 1 and then incubated with LY294002 (200 µM) for 2 h. Cells were then lysed and analysed for ABCA1, phosphorylated Akt (p-Akt) and total Akt by Western-blotting. Hsp70 was also blotted as loading control. B C ) BHK cells were labeled with [ 3 H] cholesterol and induced with 10 nM mifepristone overnight. Cholesterol efflux was measured after 2 h incubation with BSA (1 mg/ml) or BSA plus apoA-I (5 µg/ml). Some of cells were also incubated with indicated doses of LY294002 ( B ) or Akt1/2 ( C ), in addition to apoA-I, during 2 h efflux period. Results are presented as the average of triplicate wells with standard deviation and representative of more than three independent experiments. *** P

Techniques Used: Inhibition, Expressing, Incubation, Western Blot, Labeling, Standard Deviation

8) Product Images from "Caveolae Restrict Tiger Frog Virus Release in HepG2 cells and Caveolae-Associated Proteins Incorporated into Virus Particles"

Article Title: Caveolae Restrict Tiger Frog Virus Release in HepG2 cells and Caveolae-Associated Proteins Incorporated into Virus Particles

Journal: Scientific Reports

doi: 10.1038/srep21663

Virus titers of FHM cells infected with TFV. 60 h after HepG2 cells were infected with TFV at an MOI of 10. ( A ) The cells were treated with 5mM MβCD, or ( B ) 200 μg/ml nystatin for 12 h. The supernatant of the cells was collected and the Virus titers were measured. ( C ) 60 h after HepG2 cells were infected with TFV at an MOI of 10, the cells were treated with 5 mM MβCD or not (NC). The supernatant of the cells was collected at different time point (0, 2, 4, 8 and 12 h) after treated and the Virus titers were measured. Y-axes represent Virus titers. ( D ) 60 h after FHM cells were infected with TFV at an MOI of 0.1, the cells were treated with 2 mM MβCD, or 100 μg/ml nystatin for 12 h. The supernatant of the cells was collected and the virus titers were measured. Y-axes represent absolute quantitative values of TFV gene orf096r . Asterisks indicate values that are statistically significant (p
Figure Legend Snippet: Virus titers of FHM cells infected with TFV. 60 h after HepG2 cells were infected with TFV at an MOI of 10. ( A ) The cells were treated with 5mM MβCD, or ( B ) 200 μg/ml nystatin for 12 h. The supernatant of the cells was collected and the Virus titers were measured. ( C ) 60 h after HepG2 cells were infected with TFV at an MOI of 10, the cells were treated with 5 mM MβCD or not (NC). The supernatant of the cells was collected at different time point (0, 2, 4, 8 and 12 h) after treated and the Virus titers were measured. Y-axes represent Virus titers. ( D ) 60 h after FHM cells were infected with TFV at an MOI of 0.1, the cells were treated with 2 mM MβCD, or 100 μg/ml nystatin for 12 h. The supernatant of the cells was collected and the virus titers were measured. Y-axes represent absolute quantitative values of TFV gene orf096r . Asterisks indicate values that are statistically significant (p

Techniques Used: Infection

Caveolae inhibit the release of TFV virions. 60 h after HepG2 cells were infected with TFV at an MOI of 10, the cells were treated with 2, 5 and 10 mM MβCD, or 100, 200 and 500 μg/ml nystatin for 12 h. Y-axes represent absolute quantitative values of TFV gene orf096R . Asterisks indicate values that are statistically significant (p
Figure Legend Snippet: Caveolae inhibit the release of TFV virions. 60 h after HepG2 cells were infected with TFV at an MOI of 10, the cells were treated with 2, 5 and 10 mM MβCD, or 100, 200 and 500 μg/ml nystatin for 12 h. Y-axes represent absolute quantitative values of TFV gene orf096R . Asterisks indicate values that are statistically significant (p

Techniques Used: Infection

Caveolae-associated proteins were incorporated in TFV virions. Purified TFV through sedimentation in sucrose density gradients from supernatant of TFV infected HepG2 cell lines. Whole-cell lysates from the TFV-infected HepG2 (+ indicate positive control) and TFV virions underwent immunoblotting. ( A ) The blots presented in panel A were detected by viral structural proteins (MCP, ORF020R) and host proteins (CD63, CD98, alpha 1 Sodium Potassium ATPase, Glut-1). ( B ) The blots presented in panel B were detected by anti-caveolin-1, anti-caveolin-2, anti-PTRF/cavin-1, and anti-SDPR/cavin-2. ( C ) The blots presented in panel C were detected by anti-clathrin, anti-flotillin, anti-β-actin, and anti-filamin. ( D ) MβCD changed the incorporation of caveolar components. Whole-cell lysates from the TFV-infected HepG2 (+ indicate positive control) and TFV virions underwent immunoblotting. The blots presented were detected by anti-caveolin-1, anti-caveolin-2, anti-PTRF/cavin-1. ( E ) Purified TFV virions were labeled by anti-caveolin-1, anti-caveolin-2 and anti-PTRF/cavin-1 after which goat anti-rabbit IgG conjugated with 10 nm colloid gold was used for labeling the primary antibody. The NC used Negative rabbit serum as the primary antibody.
Figure Legend Snippet: Caveolae-associated proteins were incorporated in TFV virions. Purified TFV through sedimentation in sucrose density gradients from supernatant of TFV infected HepG2 cell lines. Whole-cell lysates from the TFV-infected HepG2 (+ indicate positive control) and TFV virions underwent immunoblotting. ( A ) The blots presented in panel A were detected by viral structural proteins (MCP, ORF020R) and host proteins (CD63, CD98, alpha 1 Sodium Potassium ATPase, Glut-1). ( B ) The blots presented in panel B were detected by anti-caveolin-1, anti-caveolin-2, anti-PTRF/cavin-1, and anti-SDPR/cavin-2. ( C ) The blots presented in panel C were detected by anti-clathrin, anti-flotillin, anti-β-actin, and anti-filamin. ( D ) MβCD changed the incorporation of caveolar components. Whole-cell lysates from the TFV-infected HepG2 (+ indicate positive control) and TFV virions underwent immunoblotting. The blots presented were detected by anti-caveolin-1, anti-caveolin-2, anti-PTRF/cavin-1. ( E ) Purified TFV virions were labeled by anti-caveolin-1, anti-caveolin-2 and anti-PTRF/cavin-1 after which goat anti-rabbit IgG conjugated with 10 nm colloid gold was used for labeling the primary antibody. The NC used Negative rabbit serum as the primary antibody.

Techniques Used: Purification, Sedimentation, Infection, Positive Control, Labeling

9) Product Images from "Atrial natriuretic peptide enhances microvascular albumin permeability by the caveolae-mediated transcellular pathway"

Article Title: Atrial natriuretic peptide enhances microvascular albumin permeability by the caveolae-mediated transcellular pathway

Journal: Cardiovascular Research

doi: 10.1093/cvr/cvr279

ANP, via GC-A, stimulates caveolae-mediated albumin endocytosis in MLECs. ( A ) Left, confocal images showing the uptake of Alexa 488-labelled albumin (green). The nucleus (blue) was stained with DAPI. Right, fluorescence, quantified as pixel intensity per cell using confocal microscopy, showed that ANP induced concentration-dependent increases in Alexa 488-albumin uptake by WT MLECs. These responses were abolished after pre-treatment of WT MLECs with MβCD (2 mM, 15 min; A ) as well as in GC-A-deficient ( B ) or in Cav-1-deficient MLECs ( C ). For each genotype, n = 3–4. * P
Figure Legend Snippet: ANP, via GC-A, stimulates caveolae-mediated albumin endocytosis in MLECs. ( A ) Left, confocal images showing the uptake of Alexa 488-labelled albumin (green). The nucleus (blue) was stained with DAPI. Right, fluorescence, quantified as pixel intensity per cell using confocal microscopy, showed that ANP induced concentration-dependent increases in Alexa 488-albumin uptake by WT MLECs. These responses were abolished after pre-treatment of WT MLECs with MβCD (2 mM, 15 min; A ) as well as in GC-A-deficient ( B ) or in Cav-1-deficient MLECs ( C ). For each genotype, n = 3–4. * P

Techniques Used: Aqueous Normal-phase Chromatography, Staining, Fluorescence, Confocal Microscopy, Concentration Assay

10) Product Images from "Isoprenoids determine Th1/Th2 fate in pathogenic T cells, providing a mechanism of modulation of autoimmunity by atorvastatin"

Article Title: Isoprenoids determine Th1/Th2 fate in pathogenic T cells, providing a mechanism of modulation of autoimmunity by atorvastatin

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20051129

Prenylated proteins have a regulatory function in T cell growth and differentiation. (A and B) Splenocytes taken from MBP Ac1-11 TCR Tg mice were stimulated with 5 μg/ml Ac1-11 peptide in the presence or absence of 10 μM atorvastatin (AT), 5 μM farnesyl-PP (FPP), 5 μM all-trans geranylgeranyl-PP (all-trans GGPP), 1 μM 2-cis geranylgeraniol (2-cis GGOH), or 1 μM ubiquinone (all in A) or DMSO vehicle, 5 μM of the farnesyltransferase inhibitor FTI-277 or 5 μM of the geranylgeranyltransferase-I inhibitor GGTI-298 (all in B). Proliferation (top) was measured by [ 3 H]-thymidine incorporation, and IFN-γ (middle), and IL-4 (bottom) accumulation in culture supernatants was measured via ELISA. These results are representative of data from at least three independent experiments. *, denotes a difference (P
Figure Legend Snippet: Prenylated proteins have a regulatory function in T cell growth and differentiation. (A and B) Splenocytes taken from MBP Ac1-11 TCR Tg mice were stimulated with 5 μg/ml Ac1-11 peptide in the presence or absence of 10 μM atorvastatin (AT), 5 μM farnesyl-PP (FPP), 5 μM all-trans geranylgeranyl-PP (all-trans GGPP), 1 μM 2-cis geranylgeraniol (2-cis GGOH), or 1 μM ubiquinone (all in A) or DMSO vehicle, 5 μM of the farnesyltransferase inhibitor FTI-277 or 5 μM of the geranylgeranyltransferase-I inhibitor GGTI-298 (all in B). Proliferation (top) was measured by [ 3 H]-thymidine incorporation, and IFN-γ (middle), and IL-4 (bottom) accumulation in culture supernatants was measured via ELISA. These results are representative of data from at least three independent experiments. *, denotes a difference (P

Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

The mevalonate pathway intermediate farnesyl-PP and its alcohol precursor farnesol reverse the Th2 bias promoted by atorvastatin. (A) The mevalonate pathway. Metabolites and enzymes in the pathway are shown in black, drug inhibitors are shown in red, alcohol precursors to metabolites are shown in green, and HMG-CoA reductase and pathway end-products are shown in blue. CoQ, coeznyme Q10/ubiquinone. (B) Purified naive B10.Pl CD4 + cells stimulated with 5 μg/ml αCD3/αCD28 were cultured in the presence or absence of 10 μM atorvastatin (AT), 100 μM mevalonate, or 5 μM farnesyl-PP. Proliferation (top) was measured by [ 3 H]-thymidine incorporation, and IFN-γ (middle) and IL-4 (bottom) accumulation in culture supernatants were measured by ELISA. These metabolite doses were optimal in reversal of AT effects. Values are mean ± SE of three cultures. Results are representative of three independent experiments. *, denotes a significant (P
Figure Legend Snippet: The mevalonate pathway intermediate farnesyl-PP and its alcohol precursor farnesol reverse the Th2 bias promoted by atorvastatin. (A) The mevalonate pathway. Metabolites and enzymes in the pathway are shown in black, drug inhibitors are shown in red, alcohol precursors to metabolites are shown in green, and HMG-CoA reductase and pathway end-products are shown in blue. CoQ, coeznyme Q10/ubiquinone. (B) Purified naive B10.Pl CD4 + cells stimulated with 5 μg/ml αCD3/αCD28 were cultured in the presence or absence of 10 μM atorvastatin (AT), 100 μM mevalonate, or 5 μM farnesyl-PP. Proliferation (top) was measured by [ 3 H]-thymidine incorporation, and IFN-γ (middle) and IL-4 (bottom) accumulation in culture supernatants were measured by ELISA. These metabolite doses were optimal in reversal of AT effects. Values are mean ± SE of three cultures. Results are representative of three independent experiments. *, denotes a significant (P

Techniques Used: Purification, Cell Culture, Enzyme-linked Immunosorbent Assay

11) Product Images from "The ER cholesterol sensor SCAP promotes CARTS biogenesis at ER-Golgi contact sites"

Article Title: The ER cholesterol sensor SCAP promotes CARTS biogenesis at ER-Golgi contact sites

Journal: bioRxiv

doi: 10.1101/679936

SCAP is important for PI4P turnover and VAP-A/OSBP complex distribution at ER-Golgi contact sites. a , Filipin staining in parental HeLa and shSCAP HeLa cells. High magnifications of the boxed areas are shown in the right panels. Scale bars, 10 μm. b , c , PI4P staining in parental HeLa and shSCAP HeLa cells with (top two rows in c ) or without ( b and bottom two rows in c ) sialyltransferase (ST)-mRFP expression. N, nucleus. Scale bars, 10 μm. The graph shows determination of the Golgi PI4P levels in parental HeLa (control) and shSCAP HeLa cells. Data are means ± s.e.m. (n = 139 cells per conditions, **** P
Figure Legend Snippet: SCAP is important for PI4P turnover and VAP-A/OSBP complex distribution at ER-Golgi contact sites. a , Filipin staining in parental HeLa and shSCAP HeLa cells. High magnifications of the boxed areas are shown in the right panels. Scale bars, 10 μm. b , c , PI4P staining in parental HeLa and shSCAP HeLa cells with (top two rows in c ) or without ( b and bottom two rows in c ) sialyltransferase (ST)-mRFP expression. N, nucleus. Scale bars, 10 μm. The graph shows determination of the Golgi PI4P levels in parental HeLa (control) and shSCAP HeLa cells. Data are means ± s.e.m. (n = 139 cells per conditions, **** P

Techniques Used: Staining, Expressing

12) Product Images from "Transient activation of the PI3K/Akt pathway promotes Newcastle disease virus replication and enhances anti-apoptotic signaling responses"

Article Title: Transient activation of the PI3K/Akt pathway promotes Newcastle disease virus replication and enhances anti-apoptotic signaling responses

Journal: Oncotarget

doi: 10.18632/oncotarget.15796

Inhibition of lysosomal acidification reduces Akt phosphorylation and NDV titers CEF cells were preincubated with chloroquine (5–100 μM) for 1 h and then infected with NDV-GM at an MOI of 1. Cell lysates were harvested at 1 hpi for pAkt (Ser 473), and total Akt detection was analyzed by western blot. Cell culture media supernatant aliquots were harvested at 24 hpi to determine virus titers by plaque assay. Data are representative of results of three independent experiments; ns: Not significant, * p
Figure Legend Snippet: Inhibition of lysosomal acidification reduces Akt phosphorylation and NDV titers CEF cells were preincubated with chloroquine (5–100 μM) for 1 h and then infected with NDV-GM at an MOI of 1. Cell lysates were harvested at 1 hpi for pAkt (Ser 473), and total Akt detection was analyzed by western blot. Cell culture media supernatant aliquots were harvested at 24 hpi to determine virus titers by plaque assay. Data are representative of results of three independent experiments; ns: Not significant, * p

Techniques Used: Inhibition, Infection, Western Blot, Cell Culture, Plaque Assay

Pharmacological inhibition of apoptosis enhances NDV-triggered cell survival CEF cells were pretreated with ZVAD-FMK (10, 20, 30, and 40 μM) for 1 h with or without LY294002 (20 μM) prior to infection with NDV-GM at an MOI of 1. ( A ) At 1 hpi, cells were harvested and lysed for western blot using the pAkt (Ser 473) and total Akt antibody. ( B ) At 24 hpi, cell lysates were harvested and subjected to western blot with caspase 3 and PARP antibodies. ( C ) At 24 hpi, cell viability was measured by MTT assay; * p
Figure Legend Snippet: Pharmacological inhibition of apoptosis enhances NDV-triggered cell survival CEF cells were pretreated with ZVAD-FMK (10, 20, 30, and 40 μM) for 1 h with or without LY294002 (20 μM) prior to infection with NDV-GM at an MOI of 1. ( A ) At 1 hpi, cells were harvested and lysed for western blot using the pAkt (Ser 473) and total Akt antibody. ( B ) At 24 hpi, cell lysates were harvested and subjected to western blot with caspase 3 and PARP antibodies. ( C ) At 24 hpi, cell viability was measured by MTT assay; * p

Techniques Used: Inhibition, Infection, Western Blot, MTT Assay

Inhibition of NDV-induced Akt phosphorylation following treatment with the PI3K inhibitor LY294002 (LY) ( A ) or wortmannin (wort) ( B ). CEF cells were preincubated with LY294002 (10 and 20 μM) or wort (0.2 μM and 1 μM) for 1 h, and subsequently infected with NDV-GM (a), NDV-La Sota (b), or NDV-F48E9 (c) at an MOI of 1. After 1 h, cell lysates were harvested for pAkt (Ser 473) and total Akt detection was analyzed by western blot.
Figure Legend Snippet: Inhibition of NDV-induced Akt phosphorylation following treatment with the PI3K inhibitor LY294002 (LY) ( A ) or wortmannin (wort) ( B ). CEF cells were preincubated with LY294002 (10 and 20 μM) or wort (0.2 μM and 1 μM) for 1 h, and subsequently infected with NDV-GM (a), NDV-La Sota (b), or NDV-F48E9 (c) at an MOI of 1. After 1 h, cell lysates were harvested for pAkt (Ser 473) and total Akt detection was analyzed by western blot.

Techniques Used: Inhibition, Infection, Western Blot

Early events of endocytosis on NDV infection involved in PI3K/Akt pathway activation CEF cells were preincubated with chlorpromazine (5–50 μM) ( A ) or methyl β-cyclodextrin (1–10 mM) ( B ) for 1 h and subsequently infected with NDV-GM (a), NDV-La Sota (b), or NDV-F48E9 (c) at an MOI of 1. After 1 h, cell lysates were harvested for pAkt (Ser 473), and total Akt detection was analyzed by western blot.
Figure Legend Snippet: Early events of endocytosis on NDV infection involved in PI3K/Akt pathway activation CEF cells were preincubated with chlorpromazine (5–50 μM) ( A ) or methyl β-cyclodextrin (1–10 mM) ( B ) for 1 h and subsequently infected with NDV-GM (a), NDV-La Sota (b), or NDV-F48E9 (c) at an MOI of 1. After 1 h, cell lysates were harvested for pAkt (Ser 473), and total Akt detection was analyzed by western blot.

Techniques Used: Infection, Activation Assay, Western Blot

Effect of ultraviolet (UV) irradiation on Akt activation by NDV NDV-GM with and without UV irradiation for 30 min were used to infect CEF cells, which were harvested and lysed at 0, 1, 2, and 12 hpi to detect pAkt (Ser 473) and NDV phosphoprotein (P).
Figure Legend Snippet: Effect of ultraviolet (UV) irradiation on Akt activation by NDV NDV-GM with and without UV irradiation for 30 min were used to infect CEF cells, which were harvested and lysed at 0, 1, 2, and 12 hpi to detect pAkt (Ser 473) and NDV phosphoprotein (P).

Techniques Used: Irradiation, Activation Assay

13) Product Images from "Bovine Ephemeral Fever Virus Uses a Clathrin-Mediated and Dynamin 2-Dependent Endocytosis Pathway That Requires Rab5 and Rab7 as Well as Microtubules"

Article Title: Bovine Ephemeral Fever Virus Uses a Clathrin-Mediated and Dynamin 2-Dependent Endocytosis Pathway That Requires Rab5 and Rab7 as Well as Microtubules

Journal: Journal of Virology

doi: 10.1128/JVI.01073-12

BEFV infection impaired by inhibition of dynamin 2 activity. (A) MDBK and Vero cells were pretreated with different amounts of Dynasore for 1 h and infected with BEFV at an MOI of 2. The cell lysates and supernatants of BEFV-infected cells were collected
Figure Legend Snippet: BEFV infection impaired by inhibition of dynamin 2 activity. (A) MDBK and Vero cells were pretreated with different amounts of Dynasore for 1 h and infected with BEFV at an MOI of 2. The cell lysates and supernatants of BEFV-infected cells were collected

Techniques Used: Infection, Inhibition, Activity Assay

14) Product Images from "Entry of Hepatitis B Virus into Immortalized Human Primary Hepatocytes by Clathrin-Dependent Endocytosis"

Article Title: Entry of Hepatitis B Virus into Immortalized Human Primary Hepatocytes by Clathrin-Dependent Endocytosis

Journal: Journal of Virology

doi: 10.1128/JVI.00873-12

TEM analysis of HBV-infected DMSO-treated HuS-E/2 cells and collection of secreted HBV particles. (A to C) TEM images of DMSO-treated HuS-E/2 cells producing HBV particles. Twenty hours after infection with HBV, DMSO-treated HuS-E/2 cells were washed and then incubated for 10 days, when immunogold labeling of HBsAg was performed using polyclonal anti-HBsAg antibodies, followed by secondary antibody coupled to 18-nm-diameter gold particles, and images were obtained by TEM. Black arrows, HBsAg at the ER (A) and intact HBV particles at the plasma membrane (B); white arrows, ER region in noninfected cells (C). N, nucleus; ER, endoplasmic reticulum; Cy, cytoplasm; PM, plasma membrane. (D) Western blot analysis of HBV particles secreted from HBV-infected DMSO-treated HuS-E/2 cells. DMSO-treated HuS-E/2 cells were infected with HBV for 20 h, washed with PBS, and incubated for a further 12 days. The culture medium was changed and collected every 4 days, and the samples were pooled and centrifuged to concentrate the virus particles as described in Materials and Methods. Western blot analysis was performed using anti-HBsAg antibodies. Lane −, noninfected cells as a control.
Figure Legend Snippet: TEM analysis of HBV-infected DMSO-treated HuS-E/2 cells and collection of secreted HBV particles. (A to C) TEM images of DMSO-treated HuS-E/2 cells producing HBV particles. Twenty hours after infection with HBV, DMSO-treated HuS-E/2 cells were washed and then incubated for 10 days, when immunogold labeling of HBsAg was performed using polyclonal anti-HBsAg antibodies, followed by secondary antibody coupled to 18-nm-diameter gold particles, and images were obtained by TEM. Black arrows, HBsAg at the ER (A) and intact HBV particles at the plasma membrane (B); white arrows, ER region in noninfected cells (C). N, nucleus; ER, endoplasmic reticulum; Cy, cytoplasm; PM, plasma membrane. (D) Western blot analysis of HBV particles secreted from HBV-infected DMSO-treated HuS-E/2 cells. DMSO-treated HuS-E/2 cells were infected with HBV for 20 h, washed with PBS, and incubated for a further 12 days. The culture medium was changed and collected every 4 days, and the samples were pooled and centrifuged to concentrate the virus particles as described in Materials and Methods. Western blot analysis was performed using anti-HBsAg antibodies. Lane −, noninfected cells as a control.

Techniques Used: Transmission Electron Microscopy, Infection, Incubation, Labeling, Western Blot

shRNA-mediated knockdown of CHC or AP-2 inhibits HBV infection. (A) shRNA knockdown of CHC, AP-2, or AP-1. DMSO-treated HuS-E/2 cells were transfected with plasmids expressing shRNAs specific for CHC (top), AP-2 (center), or AP-1 (bottom); cells transfected with plasmid containing shRNA against luciferase (Luc) served as controls. At 2 days posttransfection, Western blot analysis was performed using antibodies against CHC, AP-2, or AP-1, as indicated, with actin as the internal control. (B) Effect of CHC, AP-2, or AP-1 knockdown on HBV infection. DMSO-treated HuS-E/2 cells were transfected with shRNAs against CHC, AP-2, or AP-1 as described for panel A, and then at 2 days posttransfection were subjected to HBV infection or left untreated. At 12 days postinfection, RNA was isolated from the infected cells and subjected to reverse transcription and PCR analysis to detect HBV core protein mRNA. Plasmid p1.3HBcl served as the positive control. Relative expression levels of core mRNA are shown.
Figure Legend Snippet: shRNA-mediated knockdown of CHC or AP-2 inhibits HBV infection. (A) shRNA knockdown of CHC, AP-2, or AP-1. DMSO-treated HuS-E/2 cells were transfected with plasmids expressing shRNAs specific for CHC (top), AP-2 (center), or AP-1 (bottom); cells transfected with plasmid containing shRNA against luciferase (Luc) served as controls. At 2 days posttransfection, Western blot analysis was performed using antibodies against CHC, AP-2, or AP-1, as indicated, with actin as the internal control. (B) Effect of CHC, AP-2, or AP-1 knockdown on HBV infection. DMSO-treated HuS-E/2 cells were transfected with shRNAs against CHC, AP-2, or AP-1 as described for panel A, and then at 2 days posttransfection were subjected to HBV infection or left untreated. At 12 days postinfection, RNA was isolated from the infected cells and subjected to reverse transcription and PCR analysis to detect HBV core protein mRNA. Plasmid p1.3HBcl served as the positive control. Relative expression levels of core mRNA are shown.

Techniques Used: shRNA, Infection, Transfection, Expressing, Plasmid Preparation, Luciferase, Western Blot, Isolation, Polymerase Chain Reaction, Positive Control

Characterization of HuS-E/2 cells at different passages and their susceptibility to HBV. (A) Immunofluorescence staining for Ki-67. HuS-E/2 cells at passage 50 (p.50) and passage 80 (P.80) were subjected to immunofluorescence staining with rabbit anti-Ki-67 antibodies and Cy3-conjugated goat anti-rabbit IgG antibodies (red) and visualized under a fluorescence microscope (right). Phase-contrast images are shown on the left. (B) Increase in MMP-2 and MMP-9 gelatinolytic activity in HuS-E/2 cells with increased numbers of passages. The zymographic assay was performed after the indicated number of passages. (C) Levels of mRNAs coding for AFP and albumin in 293 cells, HuS-E/2 cells at passages 20, 50, and 80, HepG2 cells, and Huh7 cells. RNA was isolated from cells and analyzed by RT-PCR. Control RT-PCRs were performed for endogenous GAPDH. DNA markers are shown as molecular masses in 100-bp increments. (D) Colony formation in soft agar of HuS-E/2 cells at passage 20, 50, or 80 and of Huh7 cells. Cells were incubated in 0.35% agarose containing 10% FCS on top of 0.7% agarose containing 10% FCS at 37°C for 14 days, and then colonies were photographed under a light microscope. (E) HuS-E/2 cells at passage 50 or 80 were cultured for 12 days with 2% DMSO and incubated with or without HBV for 20 h, and then nonbound HBV was removed and the cells were incubated for an additional 12 days, when RNA was isolated and subjected to reverse transcription and PCR analysis to detect the presence of HBV HBsAg mRNA. Control PCRs were performed for endogenous GAPDH. DNA markers are shown as molecular masses in 100-bp increments.
Figure Legend Snippet: Characterization of HuS-E/2 cells at different passages and their susceptibility to HBV. (A) Immunofluorescence staining for Ki-67. HuS-E/2 cells at passage 50 (p.50) and passage 80 (P.80) were subjected to immunofluorescence staining with rabbit anti-Ki-67 antibodies and Cy3-conjugated goat anti-rabbit IgG antibodies (red) and visualized under a fluorescence microscope (right). Phase-contrast images are shown on the left. (B) Increase in MMP-2 and MMP-9 gelatinolytic activity in HuS-E/2 cells with increased numbers of passages. The zymographic assay was performed after the indicated number of passages. (C) Levels of mRNAs coding for AFP and albumin in 293 cells, HuS-E/2 cells at passages 20, 50, and 80, HepG2 cells, and Huh7 cells. RNA was isolated from cells and analyzed by RT-PCR. Control RT-PCRs were performed for endogenous GAPDH. DNA markers are shown as molecular masses in 100-bp increments. (D) Colony formation in soft agar of HuS-E/2 cells at passage 20, 50, or 80 and of Huh7 cells. Cells were incubated in 0.35% agarose containing 10% FCS on top of 0.7% agarose containing 10% FCS at 37°C for 14 days, and then colonies were photographed under a light microscope. (E) HuS-E/2 cells at passage 50 or 80 were cultured for 12 days with 2% DMSO and incubated with or without HBV for 20 h, and then nonbound HBV was removed and the cells were incubated for an additional 12 days, when RNA was isolated and subjected to reverse transcription and PCR analysis to detect the presence of HBV HBsAg mRNA. Control PCRs were performed for endogenous GAPDH. DNA markers are shown as molecular masses in 100-bp increments.

Techniques Used: Immunofluorescence, Staining, Fluorescence, Microscopy, Activity Assay, Isolation, Reverse Transcription Polymerase Chain Reaction, Incubation, Light Microscopy, Cell Culture, Polymerase Chain Reaction

HBV infection of DMSO-treated HuS-E/2 immortalized primary human hepatocytes. (A) HuS-E/2 cells were cultured for 12 days with 2% DMSO or were left untreated and were then incubated for 20 h with sera from HBV-transgenic mice (Tgm HBV infection); controls were DMSO-untreated cells not incubated with HBV (not treated [NT]). Infection was performed at a multiplicity of infection of about 5 HBV genome equivalents per cell. After removal of nonbound HBV, the cells were incubated for a further 12 days in the presence of 2% DMSO, and then RNA was isolated and amplified to detect the presence of HBV core protein mRNA. Control RT-PCRs were performed for endogenous GAPDH. The DNA markers are shown as molecular masses in 100-bp increments on the left. (B) DMSO-differentiated HuS-E/2 cells were incubated for 20 h with HBV concentrated from the culture medium of HepG2.2.15 cells grown for 12 days. Infection was performed at a multiplicity of infection of 10. RNA was then isolated and subjected to reverse transcription to generate cDNA, and PCR was performed to detect the presence of HBV HBsAg mRNA. Lane 1, PCR results for the RNA sample; lane 2, PCR results for the cDNA sample; lane 3, DNA markers; lane 4, positive control of plasmid p1.3HBcl (+). (C) Total DNA was isolated from HBV-infected DMSO-treated cells (lanes 1 and 4) or DMSO-untreated cells (lanes 2 and 5) and subjected to PCR to detect the presence of HBV cccDNA (lanes 1 and 2) or core protein DNA (lanes 4 and 5). Lanes 3 and 6, positive-control plasmid p1.3HBcl. (D) HuS-E/2 cells seeded on 18-mm coverslips were treated for 12 days with 2% DMSO or were left untreated and were then incubated with HBV for 20 h, washed, and incubated for an additional 12 days with or without 2% DMSO. Viral infection was then examined by indirect immunofluorescence staining using monoclonal anti-core protein antibody (left) and Hoechst 33258 (center), applied at the same time as the secondary antibody to label the nucleus. The stained cells were visualized by fluorescence microscopy. (E) Total DNA was isolated from the 100-fold-concentrated culture medium from HepG2.2.15 cells (lane 1), HepG2 cells (lane 2), HBV-infected DMSO-treated cells (lanes 3), or noninfected DMSO-treated cells (lane 4) and subjected to PCR to detect the HBV genomic DNA. Lanes 5 to 7, serial dilutions of a known amount of plasmid p1.3HBcl as standard.
Figure Legend Snippet: HBV infection of DMSO-treated HuS-E/2 immortalized primary human hepatocytes. (A) HuS-E/2 cells were cultured for 12 days with 2% DMSO or were left untreated and were then incubated for 20 h with sera from HBV-transgenic mice (Tgm HBV infection); controls were DMSO-untreated cells not incubated with HBV (not treated [NT]). Infection was performed at a multiplicity of infection of about 5 HBV genome equivalents per cell. After removal of nonbound HBV, the cells were incubated for a further 12 days in the presence of 2% DMSO, and then RNA was isolated and amplified to detect the presence of HBV core protein mRNA. Control RT-PCRs were performed for endogenous GAPDH. The DNA markers are shown as molecular masses in 100-bp increments on the left. (B) DMSO-differentiated HuS-E/2 cells were incubated for 20 h with HBV concentrated from the culture medium of HepG2.2.15 cells grown for 12 days. Infection was performed at a multiplicity of infection of 10. RNA was then isolated and subjected to reverse transcription to generate cDNA, and PCR was performed to detect the presence of HBV HBsAg mRNA. Lane 1, PCR results for the RNA sample; lane 2, PCR results for the cDNA sample; lane 3, DNA markers; lane 4, positive control of plasmid p1.3HBcl (+). (C) Total DNA was isolated from HBV-infected DMSO-treated cells (lanes 1 and 4) or DMSO-untreated cells (lanes 2 and 5) and subjected to PCR to detect the presence of HBV cccDNA (lanes 1 and 2) or core protein DNA (lanes 4 and 5). Lanes 3 and 6, positive-control plasmid p1.3HBcl. (D) HuS-E/2 cells seeded on 18-mm coverslips were treated for 12 days with 2% DMSO or were left untreated and were then incubated with HBV for 20 h, washed, and incubated for an additional 12 days with or without 2% DMSO. Viral infection was then examined by indirect immunofluorescence staining using monoclonal anti-core protein antibody (left) and Hoechst 33258 (center), applied at the same time as the secondary antibody to label the nucleus. The stained cells were visualized by fluorescence microscopy. (E) Total DNA was isolated from the 100-fold-concentrated culture medium from HepG2.2.15 cells (lane 1), HepG2 cells (lane 2), HBV-infected DMSO-treated cells (lanes 3), or noninfected DMSO-treated cells (lane 4) and subjected to PCR to detect the HBV genomic DNA. Lanes 5 to 7, serial dilutions of a known amount of plasmid p1.3HBcl as standard.

Techniques Used: Infection, Cell Culture, Incubation, Transgenic Assay, Mouse Assay, Isolation, Amplification, Polymerase Chain Reaction, Positive Control, Plasmid Preparation, Immunofluorescence, Staining, Fluorescence, Microscopy

Effect of BFA, CPZ, or MCD on HBV infection. (A) DMSO-treated HuS-E/2 cells were treated with 1 μM BFA, 10 μg/ml of CPZ, or 10 mM MCD for 1 h at 37°C prior to and during HBV infection for 20 h, and then the cells were washed and incubated for 12 days, when RNA was isolated and subjected to reverse transcription and PCR to detect the presence of HBV HBsAg mRNA. Control PCRs were performed for endogenous GAPDH. NT, cells without drug treatment; −, noninfected control. (B) Real-time PCR analysis of HBV gene replication in DMSO-treated HuS-E/2 hepatocytes after inhibitor treatment. The data shown are the means and standard deviations for three independent experiments. (C) Lack of effect of BFA, CPZ, or MCD on the viability of DMSO-treated HuS-E/2 cells. Cells were seeded for 24 h and then treated with 1 μM BFA, 10 μg/ml of CPZ, or 10 mM MCD for 21 h at 37°C. The cells were then washed and incubated for an additional 12 days, when cell viability was measured by the MTT assay. The number of viable cells after treatment is expressed as a percentage of that in the nontreated control (NT). The data are the mean ± standard deviation for three independent experiments.
Figure Legend Snippet: Effect of BFA, CPZ, or MCD on HBV infection. (A) DMSO-treated HuS-E/2 cells were treated with 1 μM BFA, 10 μg/ml of CPZ, or 10 mM MCD for 1 h at 37°C prior to and during HBV infection for 20 h, and then the cells were washed and incubated for 12 days, when RNA was isolated and subjected to reverse transcription and PCR to detect the presence of HBV HBsAg mRNA. Control PCRs were performed for endogenous GAPDH. NT, cells without drug treatment; −, noninfected control. (B) Real-time PCR analysis of HBV gene replication in DMSO-treated HuS-E/2 hepatocytes after inhibitor treatment. The data shown are the means and standard deviations for three independent experiments. (C) Lack of effect of BFA, CPZ, or MCD on the viability of DMSO-treated HuS-E/2 cells. Cells were seeded for 24 h and then treated with 1 μM BFA, 10 μg/ml of CPZ, or 10 mM MCD for 21 h at 37°C. The cells were then washed and incubated for an additional 12 days, when cell viability was measured by the MTT assay. The number of viable cells after treatment is expressed as a percentage of that in the nontreated control (NT). The data are the mean ± standard deviation for three independent experiments.

Techniques Used: Infection, Incubation, Isolation, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, MTT Assay, Standard Deviation

15) Product Images from "Echovirus 1 Entry into Polarized Caco-2 Cells Depends on Dynamin, Cholesterol, and Cellular Factors Associated with Macropinocytosis"

Article Title: Echovirus 1 Entry into Polarized Caco-2 Cells Depends on Dynamin, Cholesterol, and Cellular Factors Associated with Macropinocytosis

Journal: Journal of Virology

doi: 10.1128/JVI.03415-12

EV1 infection requires cholesterol. (A) Caco-2 cells were pretreated with MβCD or MβCD plus cholesterol before infection with EV1, EV7, or CVB3. (B) Cells were pretreated with MβCD before exposure to EV7 (early) or at 2 h postinfection
Figure Legend Snippet: EV1 infection requires cholesterol. (A) Caco-2 cells were pretreated with MβCD or MβCD plus cholesterol before infection with EV1, EV7, or CVB3. (B) Cells were pretreated with MβCD before exposure to EV7 (early) or at 2 h postinfection

Techniques Used: Infection

16) Product Images from "Statins stimulate the production of a soluble form of the receptor for advanced glycation end products"

Article Title: Statins stimulate the production of a soluble form of the receptor for advanced glycation end products

Journal: Journal of Lipid Research

doi: 10.1194/jlr.M038968

Influence of acute cholesterol depletion on RAGE shedding. A: Effect of cholesterol depletion on RAGE shedding in HEK/RAGE cells. Cells were incubated in the presence of 10 mM MβCD for 30, 45, and 60 min. After 4 h, the medium was collected and
Figure Legend Snippet: Influence of acute cholesterol depletion on RAGE shedding. A: Effect of cholesterol depletion on RAGE shedding in HEK/RAGE cells. Cells were incubated in the presence of 10 mM MβCD for 30, 45, and 60 min. After 4 h, the medium was collected and

Techniques Used: Incubation

17) Product Images from "Cytolethal Distending Toxin Family Members Are Differentially Affected by Alterations in Host Glycans and Membrane Cholesterol *"

Article Title: Cytolethal Distending Toxin Family Members Are Differentially Affected by Alterations in Host Glycans and Membrane Cholesterol *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.112912

CDT intoxication of CHO glycosylation mutants. A , predicted N - and O ). G M3 is the predominant glycolipid in CHO cells; therefore, the mutant structures are based on G M3 . B , cells were intoxicated
Figure Legend Snippet: CDT intoxication of CHO glycosylation mutants. A , predicted N - and O ). G M3 is the predominant glycolipid in CHO cells; therefore, the mutant structures are based on G M3 . B , cells were intoxicated

Techniques Used: Mutagenesis

18) Product Images from "Beyond the redox imbalance: oxidative stress contributes to an impaired GLUT3 modulation in Huntington's disease"

Article Title: Beyond the redox imbalance: oxidative stress contributes to an impaired GLUT3 modulation in Huntington's disease

Journal: Free radical biology & medicine

doi: 10.1016/j.freeradbiomed.2015.09.024

GLUT3 behaviour at the plasma membrane does not change in HD cells (a) Immnufluorescence analyses in Q7 and Q111 cells for GLUT3 (green). Direct fluorescence corresponding to GLUT3-mCherry is shown in red. Line-scan data for yellow lines drawn across cells in the merged panels is shown. (b) TIRF microscopy images for representative Q7 and Q111 cells expressing GLUT3-EGFP. Cells were treated with cholesterol (30 µg/ml) or methyl-β-cyclodextrin (MβCD: 10 mM) for the time indicated. Bar plots for normalised fluorescence intensity obtained from TIRF microscopy images in Q cells expressing GLUT3-EGFP. Analysis of variance (ANOVA) followed by the Bonferroni post-test, n=3.
Figure Legend Snippet: GLUT3 behaviour at the plasma membrane does not change in HD cells (a) Immnufluorescence analyses in Q7 and Q111 cells for GLUT3 (green). Direct fluorescence corresponding to GLUT3-mCherry is shown in red. Line-scan data for yellow lines drawn across cells in the merged panels is shown. (b) TIRF microscopy images for representative Q7 and Q111 cells expressing GLUT3-EGFP. Cells were treated with cholesterol (30 µg/ml) or methyl-β-cyclodextrin (MβCD: 10 mM) for the time indicated. Bar plots for normalised fluorescence intensity obtained from TIRF microscopy images in Q cells expressing GLUT3-EGFP. Analysis of variance (ANOVA) followed by the Bonferroni post-test, n=3.

Techniques Used: Fluorescence, Microscopy, Expressing

19) Product Images from "Lack of Fusion of Azurophil Granules with Phagosomes during Phagocytosis of Mycobacterium smegmatis by Human Neutrophils Is Not Actively Controlled by the Bacterium"

Article Title: Lack of Fusion of Azurophil Granules with Phagosomes during Phagocytosis of Mycobacterium smegmatis by Human Neutrophils Is Not Actively Controlled by the Bacterium

Journal: Infection and Immunity

doi: 10.1128/IAI.70.3.1591-1598.2002

Fusion of azurophil granules with phagosomes in neutrophils is triggered by IgG opsonization of M. smegmatis. (A) Neutrophils were incubated with immune serum-opsonized zymosan (OZ) (20 particles per cell) for 20 min or with live, heat-killed or immune serum-opsonized M. smegmatis for 40 min (50 particle per cell). The release of the lysosomal enzyme β-glucuronidase was measured. Results are expressed as means + SDs (error bars) of three experiments. ∗∗∗, P
Figure Legend Snippet: Fusion of azurophil granules with phagosomes in neutrophils is triggered by IgG opsonization of M. smegmatis. (A) Neutrophils were incubated with immune serum-opsonized zymosan (OZ) (20 particles per cell) for 20 min or with live, heat-killed or immune serum-opsonized M. smegmatis for 40 min (50 particle per cell). The release of the lysosomal enzyme β-glucuronidase was measured. Results are expressed as means + SDs (error bars) of three experiments. ∗∗∗, P

Techniques Used: Incubation

20) Product Images from "Fate of cerium dioxide nanoparticles in endothelial cells: exocytosis"

Article Title: Fate of cerium dioxide nanoparticles in endothelial cells: exocytosis

Journal: Journal of Nanoparticle Research

doi: 10.1007/s11051-015-3007-4

Decrease of intracellular nanoparticle accumulation and partial re-uptake of exocytosed nanoparticles with increasing time after exposure. a With increasing follow-up time after nanoparticle exposure a continuous decrease of the intracellular fluorescence intensity was observed. This indicates a cellular nanoparticle decrease as result of exocytosis and cell division (“nanoparticle dilution”). n = 3 independent experiments; MFI median fluorescence intensity of the cell population; asterisks indicate significant differences ( P ≤ 0.05) to the initial value (“0 h” follow-up; 100 %), different letters indicate significant differences ( P ≤ 0.05) between different time points. b The occurrence of cerium (Ce) in the supernatant of endothelial cells, which were previously exposed to CeO 2 nanoparticles and which were followed up after washing and cell culture medium exchange (nanoparticle free medium), revealed the occurrence of exocytosis of intracellular nanoparticles. The lower Ce supernatant concentrations which were found with increasing follow-up time (48 and 72-h follow-up time) in comparison to 24-h follow-up time indicate a re-uptake of exocytosed nanoparticles in cells. The Ce content in supernatants of cells, which were not treated with nanoparticles, was below the detection limit. n = 2 independent experiments
Figure Legend Snippet: Decrease of intracellular nanoparticle accumulation and partial re-uptake of exocytosed nanoparticles with increasing time after exposure. a With increasing follow-up time after nanoparticle exposure a continuous decrease of the intracellular fluorescence intensity was observed. This indicates a cellular nanoparticle decrease as result of exocytosis and cell division (“nanoparticle dilution”). n = 3 independent experiments; MFI median fluorescence intensity of the cell population; asterisks indicate significant differences ( P ≤ 0.05) to the initial value (“0 h” follow-up; 100 %), different letters indicate significant differences ( P ≤ 0.05) between different time points. b The occurrence of cerium (Ce) in the supernatant of endothelial cells, which were previously exposed to CeO 2 nanoparticles and which were followed up after washing and cell culture medium exchange (nanoparticle free medium), revealed the occurrence of exocytosis of intracellular nanoparticles. The lower Ce supernatant concentrations which were found with increasing follow-up time (48 and 72-h follow-up time) in comparison to 24-h follow-up time indicate a re-uptake of exocytosed nanoparticles in cells. The Ce content in supernatants of cells, which were not treated with nanoparticles, was below the detection limit. n = 2 independent experiments

Techniques Used: Fluorescence, Cell Culture

Strong inhibitory effect of MβcD and brefeldin A indicates the important role of plasma membrane cholesterol and Golgi-to-cell-surface-transport, respectively, during nanoparticles exocytosis. a The average exocytosis rate of CeO 2 nanoparticles (treatment dose 1 µg/ml for 24 h) within 24 h was 62 ± 5 %. Nocodazole led to no obvious inhibition of nanoparticle exocytosis (exocytosis rate: 59 ± 2 %). The highest inhibition of exocytosis was caused by MβcD with an exposure time of 2 h indicating an important role of plasma membrane cholesterol for exocytosis. Brefeldin A treatment resulted also in an inhibition of exocytosis revealing an involvement of Golgi-to-cell-surface-transport in exocytosis process. Different letters indicate significant differences ( P ≤ 0.05) between the various treatments. n ≥ 3 independent experiments; b Histograms of a representative flow cytometry analysis; NPs: nanoparticles. c The determination of cerium (Ce) in the supernatant of HMEC-1, which were exposed to nanoparticles for 24 h, revealed 24 h after washing and cell culture medium exchange (nanoparticle free medium) a higher amount of Ce than the supernatants of HMEC-1 which were additionally treated with MβcD-containing cell culture medium after washing and medium exchange. This confirmed the inhibition of exocytosis by MβcD. The Ce content in supernatants of cells, which were not treated with nanoparticles, was below the detection limit. n = 2 independent experiments
Figure Legend Snippet: Strong inhibitory effect of MβcD and brefeldin A indicates the important role of plasma membrane cholesterol and Golgi-to-cell-surface-transport, respectively, during nanoparticles exocytosis. a The average exocytosis rate of CeO 2 nanoparticles (treatment dose 1 µg/ml for 24 h) within 24 h was 62 ± 5 %. Nocodazole led to no obvious inhibition of nanoparticle exocytosis (exocytosis rate: 59 ± 2 %). The highest inhibition of exocytosis was caused by MβcD with an exposure time of 2 h indicating an important role of plasma membrane cholesterol for exocytosis. Brefeldin A treatment resulted also in an inhibition of exocytosis revealing an involvement of Golgi-to-cell-surface-transport in exocytosis process. Different letters indicate significant differences ( P ≤ 0.05) between the various treatments. n ≥ 3 independent experiments; b Histograms of a representative flow cytometry analysis; NPs: nanoparticles. c The determination of cerium (Ce) in the supernatant of HMEC-1, which were exposed to nanoparticles for 24 h, revealed 24 h after washing and cell culture medium exchange (nanoparticle free medium) a higher amount of Ce than the supernatants of HMEC-1 which were additionally treated with MβcD-containing cell culture medium after washing and medium exchange. This confirmed the inhibition of exocytosis by MβcD. The Ce content in supernatants of cells, which were not treated with nanoparticles, was below the detection limit. n = 2 independent experiments

Techniques Used: Inhibition, Flow Cytometry, Cytometry, Cell Culture

Intracellular localization of nanoparticles with increasing time after exposure and after inhibitor application. TEM images of HEMC-1 after treatment with 10 µg/ml CeO 2 nanoparticles for 24 h show endocytosis ( a ) and the internalized nanoparticles in endosomes ( b ) as well as in the cytosol ( c ). TEM images of the localization of the nanoparticles 24 h after washing and medium exchange with nanoparticle free medium ( d – i ) revealed clearly the initiation ( e , f ) and occurrence ( h , i ) of exocytosis of the internalized nanoparticles. Cells treated with brefeldin A ( j , k ; 0.1 µg/ml, 24 h), nocodazole ( l , m ; 10 µg/ml, 24 h), or MβcD ( n , o ; 10 mM, 2 h) revealed no or hardly any exocytosis ( m ), but the localization of nanoparticles in large endosomes ( j , l , n ) or in the cytoplasm ( k , m , o ). The occurrence of nanoparticles in the cytoplasm indicated endosomal perforation. Arrows ( f , i , m ) point to the cytosolic nanoparticles which are shortly before exocytosis. Scale bars : 0.1 ( m ); 0.3 µm ( b , c , d , f ); 0.5 µm ( i ); 1.0 µm ( e , g , j , k , n ); 1.5 µm ( a , h ); 2.0 ( l , o )
Figure Legend Snippet: Intracellular localization of nanoparticles with increasing time after exposure and after inhibitor application. TEM images of HEMC-1 after treatment with 10 µg/ml CeO 2 nanoparticles for 24 h show endocytosis ( a ) and the internalized nanoparticles in endosomes ( b ) as well as in the cytosol ( c ). TEM images of the localization of the nanoparticles 24 h after washing and medium exchange with nanoparticle free medium ( d – i ) revealed clearly the initiation ( e , f ) and occurrence ( h , i ) of exocytosis of the internalized nanoparticles. Cells treated with brefeldin A ( j , k ; 0.1 µg/ml, 24 h), nocodazole ( l , m ; 10 µg/ml, 24 h), or MβcD ( n , o ; 10 mM, 2 h) revealed no or hardly any exocytosis ( m ), but the localization of nanoparticles in large endosomes ( j , l , n ) or in the cytoplasm ( k , m , o ). The occurrence of nanoparticles in the cytoplasm indicated endosomal perforation. Arrows ( f , i , m ) point to the cytosolic nanoparticles which are shortly before exocytosis. Scale bars : 0.1 ( m ); 0.3 µm ( b , c , d , f ); 0.5 µm ( i ); 1.0 µm ( e , g , j , k , n ); 1.5 µm ( a , h ); 2.0 ( l , o )

Techniques Used: Transmission Electron Microscopy

Nanoparticles’ features. a TEM pictures of the CeO 2 nanoparticles show the varying shapes. b Structure of the reactive dye species ATTO 647 N-APS used for labeling. c Stability test of the ATTO 647 N-APS label in endothelial cell culture medium (PAA Laboratories, Pasching, Austria) revealed the stability of the dye for at least 2 weeks, since 90–95 % of the initial fluorescence intensity of the ATTO 647 N-APS label was present after this period
Figure Legend Snippet: Nanoparticles’ features. a TEM pictures of the CeO 2 nanoparticles show the varying shapes. b Structure of the reactive dye species ATTO 647 N-APS used for labeling. c Stability test of the ATTO 647 N-APS label in endothelial cell culture medium (PAA Laboratories, Pasching, Austria) revealed the stability of the dye for at least 2 weeks, since 90–95 % of the initial fluorescence intensity of the ATTO 647 N-APS label was present after this period

Techniques Used: Transmission Electron Microscopy, Labeling, Cell Culture, Fluorescence

21) Product Images from "Mild Lipid Stress Induces Profound Loss of MC4R Protein Abundance and Function"

Article Title: Mild Lipid Stress Induces Profound Loss of MC4R Protein Abundance and Function

Journal: Molecular Endocrinology

doi: 10.1210/me.2013-1357

Elevated palmitate induces profound loss of cAMP signaling in response to α-MSH in N42 hypothalamic neurons and immortalized N2A HA-MC4R-GFP cells. A, N42 cells were treated without or with palmitate at the indicated concentrations for 24 hours. Cells were collected for mRNA isolation and quantitative PCR performed using primers specific to MC4R and actin. Data are shown as the amount of MC4R transcript normalized to that of actin. B, N42 cells were treated with palmitate as in A and then stimulated with 100 nM α-MSH or 1 μM forskolin. Samples were collected to measure cell cAMP levels and protein concentration. C, N2A HA-MC4R-GFP cells were treated with palmitate and the amount of MC4R and HA-MC4R-GFP transcripts, normalized to that of actin, was determined as in A. D, N2A HA-MC4R-GFP cells were treated with palmitate as in A, and cAMP and protein concentration was determined. Data are expressed as mean ± SD. **, P
Figure Legend Snippet: Elevated palmitate induces profound loss of cAMP signaling in response to α-MSH in N42 hypothalamic neurons and immortalized N2A HA-MC4R-GFP cells. A, N42 cells were treated without or with palmitate at the indicated concentrations for 24 hours. Cells were collected for mRNA isolation and quantitative PCR performed using primers specific to MC4R and actin. Data are shown as the amount of MC4R transcript normalized to that of actin. B, N42 cells were treated with palmitate as in A and then stimulated with 100 nM α-MSH or 1 μM forskolin. Samples were collected to measure cell cAMP levels and protein concentration. C, N2A HA-MC4R-GFP cells were treated with palmitate and the amount of MC4R and HA-MC4R-GFP transcripts, normalized to that of actin, was determined as in A. D, N2A HA-MC4R-GFP cells were treated with palmitate as in A, and cAMP and protein concentration was determined. Data are expressed as mean ± SD. **, P

Techniques Used: Isolation, Real-time Polymerase Chain Reaction, Protein Concentration

22) Product Images from "Rab5, Rab7, and Rab11 Are Required for Caveola-Dependent Endocytosis of Classical Swine Fever Virus in Porcine Alveolar Macrophages"

Article Title: Rab5, Rab7, and Rab11 Are Required for Caveola-Dependent Endocytosis of Classical Swine Fever Virus in Porcine Alveolar Macrophages

Journal: Journal of Virology

doi: 10.1128/JVI.00797-18

CSFV entry requires acidic endosomal pH. (A to C) Chloroquine, NH 4 Cl, and BafA1 inhibited CSFV entry. Cells were pretreated with subtoxic doses at 37°C for 1 h and inoculated with CSFV (MOI = 1) at 4°C for 1 h and then shifted to 37°C. At 0 (binding) or 1 (entry) hpi, infected cells were lysed to determine the viral RNA copy number by RT-qPCR. (D) V-ATPase knockdown inhibited CSFV infection. siV-ATPase or siCtrl-transfected cells were infected with CSFV (MOI = 0.1). At 24 hpi, the viral RNA levels, virus titers, and viral E2 protein levels were determined by RT-qPCR, virus titration, and Western blotting, respectively. The relative protein amount was calculated using ImageJ 7.0 software as shown in the figure. The results are presented as means ± the SD of data from three independent experiments (**, P
Figure Legend Snippet: CSFV entry requires acidic endosomal pH. (A to C) Chloroquine, NH 4 Cl, and BafA1 inhibited CSFV entry. Cells were pretreated with subtoxic doses at 37°C for 1 h and inoculated with CSFV (MOI = 1) at 4°C for 1 h and then shifted to 37°C. At 0 (binding) or 1 (entry) hpi, infected cells were lysed to determine the viral RNA copy number by RT-qPCR. (D) V-ATPase knockdown inhibited CSFV infection. siV-ATPase or siCtrl-transfected cells were infected with CSFV (MOI = 0.1). At 24 hpi, the viral RNA levels, virus titers, and viral E2 protein levels were determined by RT-qPCR, virus titration, and Western blotting, respectively. The relative protein amount was calculated using ImageJ 7.0 software as shown in the figure. The results are presented as means ± the SD of data from three independent experiments (**, P

Techniques Used: Binding Assay, Infection, Quantitative RT-PCR, Transfection, Titration, Western Blot, Software

23) Product Images from "Modulation of Cellular Cholesterol Transport and Homeostasis by Rab11"

Article Title: Modulation of Cellular Cholesterol Transport and Homeostasis by Rab11

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E02-01-0025

Distribution of free cholesterol in COS-1 cells. Left, cells were stained with FITC-conjugated lentil lectin, anti-lamp1 antibodies, biotin-2xFYVE, and Cy3-conjugated streptavidin or anti-Rab11 antibodies. Right, filipin stainings of the respective cells. The arrowheads indicate colocalization of endosomal markers with filipin staining. Images were obtained using wide-field microscope. Bar, 8 μm.
Figure Legend Snippet: Distribution of free cholesterol in COS-1 cells. Left, cells were stained with FITC-conjugated lentil lectin, anti-lamp1 antibodies, biotin-2xFYVE, and Cy3-conjugated streptavidin or anti-Rab11 antibodies. Right, filipin stainings of the respective cells. The arrowheads indicate colocalization of endosomal markers with filipin staining. Images were obtained using wide-field microscope. Bar, 8 μm.

Techniques Used: Staining, Microscopy

24) Product Images from "Lateral Diffusion, Function, and Expression of the Slow Channel Congenital Myasthenia Syndrome αC418W Nicotinic Receptor Mutation with Changes in Lipid Raft Components *"

Article Title: Lateral Diffusion, Function, and Expression of the Slow Channel Congenital Myasthenia Syndrome αC418W Nicotinic Receptor Mutation with Changes in Lipid Raft Components *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M115.678573

Modulation of cholesterol content in HEK 293 cells transfected with the nAChR. Membrane cholesterol levels of HEK 293 cells expressing the WT and αC418W mutant nAChR after cholesterol enrichment or depletion.
Figure Legend Snippet: Modulation of cholesterol content in HEK 293 cells transfected with the nAChR. Membrane cholesterol levels of HEK 293 cells expressing the WT and αC418W mutant nAChR after cholesterol enrichment or depletion.

Techniques Used: Transfection, Expressing, Mutagenesis

Lateral Mobility of the WT and the αC418W Mutant nAChR at the Cell Surface of HEK 293 Cells
Figure Legend Snippet: Lateral Mobility of the WT and the αC418W Mutant nAChR at the Cell Surface of HEK 293 Cells

Techniques Used: Mutagenesis

25) Product Images from "Activation of integrin α5 mediated by flow requires its translocation to membrane lipid rafts in vascular endothelial cells"

Article Title: Activation of integrin α5 mediated by flow requires its translocation to membrane lipid rafts in vascular endothelial cells

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

doi: 10.1073/pnas.1524523113

( A ) ECs pretreated with or without 2.5 μmol/L Cyt-B for 2 h were subjected to OS for 2 h. Act-α5 and total-α5 (α5) in lipid rafts (Rafts) were measured. ( B and C ) Bar graphs show the relative ratio of act-α5 ( B
Figure Legend Snippet: ( A ) ECs pretreated with or without 2.5 μmol/L Cyt-B for 2 h were subjected to OS for 2 h. Act-α5 and total-α5 (α5) in lipid rafts (Rafts) were measured. ( B and C ) Bar graphs show the relative ratio of act-α5 ( B

Techniques Used: Activated Clotting Time Assay

26) Product Images from "Sphingolipids affect fibrinogen-induced caveolar transcytosis and cerebrovascular permeability"

Article Title: Sphingolipids affect fibrinogen-induced caveolar transcytosis and cerebrovascular permeability

Journal: American Journal of Physiology - Cell Physiology

doi: 10.1152/ajpcell.00305.2013

Fibrinogen (Fg)-induced permeability of mouse brain endothelial cells (MBECs). A and B : permeability of MBECs to Lucifer yellow (LY) and BSA tagged with Alexa Fluor 647 (BSA-647) in the presence of PBS in medium (control), 4 mg/ml fibrinogen (Fg4), 4 mg/ml Fg + 100 μM methyl-β-cyclodextrin (Fg4 + MβCD), or 100 μM MβCD. C and D : permeability of MBECs to LY and BSA-647 in the presence of PBS in medium (control), 4 mg/ml Fg (Fg4), 4 mg/ml Fg + 500 nM myriocin (Fg4 + myriocin), or 500 nM myriocin. Fluorescence intensity of each dye in samples collected from lower chambers of the Transwell system after 20, 40, 60, and 120 min was measured by a microplate reader (488-nm excitation and 520-nm emission for LY; 650-nm excitation and 668-nm emission for BSA-647). Results are expressed as ratio of fluorescence intensity of each dye in the lower chamber to fluorescence intensity of the respective dye in the original sample at the end of the experiment. Values are means ± SE; n = 4. * P
Figure Legend Snippet: Fibrinogen (Fg)-induced permeability of mouse brain endothelial cells (MBECs). A and B : permeability of MBECs to Lucifer yellow (LY) and BSA tagged with Alexa Fluor 647 (BSA-647) in the presence of PBS in medium (control), 4 mg/ml fibrinogen (Fg4), 4 mg/ml Fg + 100 μM methyl-β-cyclodextrin (Fg4 + MβCD), or 100 μM MβCD. C and D : permeability of MBECs to LY and BSA-647 in the presence of PBS in medium (control), 4 mg/ml Fg (Fg4), 4 mg/ml Fg + 500 nM myriocin (Fg4 + myriocin), or 500 nM myriocin. Fluorescence intensity of each dye in samples collected from lower chambers of the Transwell system after 20, 40, 60, and 120 min was measured by a microplate reader (488-nm excitation and 520-nm emission for LY; 650-nm excitation and 668-nm emission for BSA-647). Results are expressed as ratio of fluorescence intensity of each dye in the lower chamber to fluorescence intensity of the respective dye in the original sample at the end of the experiment. Values are means ± SE; n = 4. * P

Techniques Used: Permeability, Fluorescence

27) Product Images from "Opposing Effects of Dopamine D1- and D2-Like Agonists on Intracranial Self-Stimulation in Male Rats"

Article Title: Opposing Effects of Dopamine D1- and D2-Like Agonists on Intracranial Self-Stimulation in Male Rats

Journal: Experimental and clinical psychopharmacology

doi: 10.1037/pha0000067

Effects of the D2/3 agonists (A) sumanirole (B) apomorphine (C) quinpirole (D) PD128907 (E) pramipexole and (F) aripiprazole
Figure Legend Snippet: Effects of the D2/3 agonists (A) sumanirole (B) apomorphine (C) quinpirole (D) PD128907 (E) pramipexole and (F) aripiprazole

Techniques Used:

28) Product Images from "CD44 ligation with A3D8 antibody induces apoptosis in acute myeloid leukemia cells through binding to CD44s and clustering lipid rafts"

Article Title: CD44 ligation with A3D8 antibody induces apoptosis in acute myeloid leukemia cells through binding to CD44s and clustering lipid rafts

Journal: Cancer Biology & Therapy

doi: 10.4161/cbt.21784

HMWHA and J173 neither induce apoptosis nor induce clustering of lipid rafts in NB4 cells. NB4 cells were treated with HMWHA 350 μg/ml, J173 2.5 μg/ml, A3D8 2.5 μg/ml and dialyzed A3D8 (A3D8-D) 2.5 μg/ml for 72 h. The percentage of apoptotic cells was determined by FACS after staining with annexin-V (A). Lipid raft clustering was determined by confocal microscopy after staining with the FITC-CtxB subunit to identify lipid rafts (green fluorescence) and to identify nuclei by staining with DAPI (B).
Figure Legend Snippet: HMWHA and J173 neither induce apoptosis nor induce clustering of lipid rafts in NB4 cells. NB4 cells were treated with HMWHA 350 μg/ml, J173 2.5 μg/ml, A3D8 2.5 μg/ml and dialyzed A3D8 (A3D8-D) 2.5 μg/ml for 72 h. The percentage of apoptotic cells was determined by FACS after staining with annexin-V (A). Lipid raft clustering was determined by confocal microscopy after staining with the FITC-CtxB subunit to identify lipid rafts (green fluorescence) and to identify nuclei by staining with DAPI (B).

Techniques Used: FACS, Staining, Confocal Microscopy, Fluorescence

Disruption of lipid rafts with MCD abrogates A3D8-induced apoptosis in NB4 cells. NB4 cells were treated with A3D8 at 2.5 μg/ml for 2 days and then with 2.5 mg/ml MCD for 30 min. MCD was washed out and cells were treated with or without A3D8 2.5 μg/ml for another 24 h. Lipid rafts were determined with a confocal microscopy (A). Cells were fixed and stained with the FITC-Ctx B subunit to identify rafts (green fluorescence) and nuclei were identified by staining with DAPI. The percentage of apoptotic cells in NB cells treated with A3D8 and/or MCD was measured by FACS after staining with Annexin-V (B). The relative levels of cleaved caspase-3, -8 and PARP in NB cells treated with A3D8 and/or MCD were analyzed by Western blotting (C).
Figure Legend Snippet: Disruption of lipid rafts with MCD abrogates A3D8-induced apoptosis in NB4 cells. NB4 cells were treated with A3D8 at 2.5 μg/ml for 2 days and then with 2.5 mg/ml MCD for 30 min. MCD was washed out and cells were treated with or without A3D8 2.5 μg/ml for another 24 h. Lipid rafts were determined with a confocal microscopy (A). Cells were fixed and stained with the FITC-Ctx B subunit to identify rafts (green fluorescence) and nuclei were identified by staining with DAPI. The percentage of apoptotic cells in NB cells treated with A3D8 and/or MCD was measured by FACS after staining with Annexin-V (B). The relative levels of cleaved caspase-3, -8 and PARP in NB cells treated with A3D8 and/or MCD were analyzed by Western blotting (C).

Techniques Used: Confocal Microscopy, Staining, Fluorescence, FACS, Western Blot

Fas is clustered into membrane lipid rafts in NB4 cells after A3D8 treatment. NB4 cells were treated with or without 2.5 μg/ml A3D8 or mouse IgG for 72 h. The cells were fixed and stained with the FITC-Ctx B subunit to identify lipid rafts (green fluorescence) and with an anti-Fas antibody to identify Fas (red fluorescence). Areas of colocalization between membrane rafts and Fas are yellow.
Figure Legend Snippet: Fas is clustered into membrane lipid rafts in NB4 cells after A3D8 treatment. NB4 cells were treated with or without 2.5 μg/ml A3D8 or mouse IgG for 72 h. The cells were fixed and stained with the FITC-Ctx B subunit to identify lipid rafts (green fluorescence) and with an anti-Fas antibody to identify Fas (red fluorescence). Areas of colocalization between membrane rafts and Fas are yellow.

Techniques Used: Staining, Fluorescence

A3D8 treatment induces apoptosis in NB4 cells through activation of caspase-8. (A) Apoptosis induction. NB4 cells were treated with A3D8 at the indicated concentrations for 1 to 3 days. The percentage of apoptotic cells were determined by FACS after staining with annexin-V. The data shown are the mean plus SE of three independent experiments. (B) The levels of cleaved PARP, caspase-3, -8 and -9. NB4 cells were treated with 2.5 μg/ml A3D8 for 1 to 3 days and the relative levels of the indicated proteins were analyzed by Western blotting using specific antibodies. GAPDH levels were used as loading controls. (C) Inhibition of A3D8-induced apoptosis by caspase inhibitors. NB4 cells were pretreated with the pancaspase inhibitor Z-VAD (50 μM), the caspase-9 inhibitor Z-LETD (50 μM), the caspase-8 inhibitor Z-IETD (50 μM) for 4 h and then with 2.5 μg/ml A3D8 for 72 h. The percentage of apoptotic cells were detected by FACS after staining with annexin V. The data shown are the mean plus SE of three independent experiments.
Figure Legend Snippet: A3D8 treatment induces apoptosis in NB4 cells through activation of caspase-8. (A) Apoptosis induction. NB4 cells were treated with A3D8 at the indicated concentrations for 1 to 3 days. The percentage of apoptotic cells were determined by FACS after staining with annexin-V. The data shown are the mean plus SE of three independent experiments. (B) The levels of cleaved PARP, caspase-3, -8 and -9. NB4 cells were treated with 2.5 μg/ml A3D8 for 1 to 3 days and the relative levels of the indicated proteins were analyzed by Western blotting using specific antibodies. GAPDH levels were used as loading controls. (C) Inhibition of A3D8-induced apoptosis by caspase inhibitors. NB4 cells were pretreated with the pancaspase inhibitor Z-VAD (50 μM), the caspase-9 inhibitor Z-LETD (50 μM), the caspase-8 inhibitor Z-IETD (50 μM) for 4 h and then with 2.5 μg/ml A3D8 for 72 h. The percentage of apoptotic cells were detected by FACS after staining with annexin V. The data shown are the mean plus SE of three independent experiments.

Techniques Used: Activation Assay, FACS, Staining, Western Blot, Inhibition

A3D8 and J173 antibodies have different binding abilities to HL-60, SKNO-1 and NB4 cells. (A) Western blot analysis of CD44 protein levels. Cellular lysates were isolated from HL-60, SKNO-1 and NB4 cells, subjected to 8% SDS-gel electrophoresis and then probed with either A3D8 or J173 antibody. (B) Cell surface CD44 binding of A3D8 and J173. HL-60, SKNO-1 and NB4 cells were incubated with mouse IgG, A3D8 and J173 first and then FITC labeled secondary antibody. The fluorescence strength was determined by FACS.
Figure Legend Snippet: A3D8 and J173 antibodies have different binding abilities to HL-60, SKNO-1 and NB4 cells. (A) Western blot analysis of CD44 protein levels. Cellular lysates were isolated from HL-60, SKNO-1 and NB4 cells, subjected to 8% SDS-gel electrophoresis and then probed with either A3D8 or J173 antibody. (B) Cell surface CD44 binding of A3D8 and J173. HL-60, SKNO-1 and NB4 cells were incubated with mouse IgG, A3D8 and J173 first and then FITC labeled secondary antibody. The fluorescence strength was determined by FACS.

Techniques Used: Binding Assay, Western Blot, Isolation, SDS-Gel, Electrophoresis, Incubation, Labeling, Fluorescence, FACS

SKNO-1 cells, but not HL-60 cells, are sensitive to A3D8-induced apoptosis and caspase-8 activation. SKNO-1 cells were treated with or without 2.5 μg/ml A3D8 for 2 days and HL-60 cells were treated with or without 2.5 μg/ml A3D8 for 3 days. Apoptotic cells were determined by FACS after staining with annexin-V (A). The relative levels of cleaved PARP, caspase-3 and -8 were analyzed by Western blotting analysis (B).
Figure Legend Snippet: SKNO-1 cells, but not HL-60 cells, are sensitive to A3D8-induced apoptosis and caspase-8 activation. SKNO-1 cells were treated with or without 2.5 μg/ml A3D8 for 2 days and HL-60 cells were treated with or without 2.5 μg/ml A3D8 for 3 days. Apoptotic cells were determined by FACS after staining with annexin-V (A). The relative levels of cleaved PARP, caspase-3 and -8 were analyzed by Western blotting analysis (B).

Techniques Used: Activation Assay, FACS, Staining, Western Blot

29) Product Images from "The ?7 nicotinic acetylcholine receptor function in hippocampal neurons is regulated by the lipid composition of the plasma membrane"

Article Title: The ?7 nicotinic acetylcholine receptor function in hippocampal neurons is regulated by the lipid composition of the plasma membrane

Journal: The Journal of Physiology

doi: 10.1113/jphysiol.2011.209494

Cholesterol and sphingomyelin prevent the effects of lipid raft disruption through MβCD and SMase on desensitization kinetics of neuronal α7 nAChRs
Figure Legend Snippet: Cholesterol and sphingomyelin prevent the effects of lipid raft disruption through MβCD and SMase on desensitization kinetics of neuronal α7 nAChRs

Techniques Used:

30) Product Images from "Cholesterol Organization in Phosphatidylcholine Liposomes: A Surface Plasmon Resonance Study"

Article Title: Cholesterol Organization in Phosphatidylcholine Liposomes: A Surface Plasmon Resonance Study

Journal: Materials

doi: 10.3390/ma5112306

The exponential decay time constants for cholesterol removal from the liposomes are shown as a function of the initial cholesterol mole fraction in liposomes made from OPPC (●) and POPC (○). The time constants ( t ) refer to the single exponential decay observed on addition of βCD to adsorbed liposomes; larger values for t indicate slower removal of the cholesterol. The time constants are also shown here for liposomes saturated with cholesterol using MβCD: Chl; values are for the slow pool of cholesterol, as described below. The half-life for cholesterol in the sample is given by t ln2, and therefore ranges here from 100 to 330 s.
Figure Legend Snippet: The exponential decay time constants for cholesterol removal from the liposomes are shown as a function of the initial cholesterol mole fraction in liposomes made from OPPC (●) and POPC (○). The time constants ( t ) refer to the single exponential decay observed on addition of βCD to adsorbed liposomes; larger values for t indicate slower removal of the cholesterol. The time constants are also shown here for liposomes saturated with cholesterol using MβCD: Chl; values are for the slow pool of cholesterol, as described below. The half-life for cholesterol in the sample is given by t ln2, and therefore ranges here from 100 to 330 s.

Techniques Used:

Representative raw data for a cycle of liposome deposition ( χ C = 0.35), MβCD: Chl addition and βCD addition on the modified surface of the SPR slide. The addition times for these solutions are indicated by arrows on the Figure. The βCD removes both the cholesterol added via the MβCD: Chl and the cholesterol initially present, so that the final signal before the detergent rinse is lower than the initial signal.
Figure Legend Snippet: Representative raw data for a cycle of liposome deposition ( χ C = 0.35), MβCD: Chl addition and βCD addition on the modified surface of the SPR slide. The addition times for these solutions are indicated by arrows on the Figure. The βCD removes both the cholesterol added via the MβCD: Chl and the cholesterol initially present, so that the final signal before the detergent rinse is lower than the initial signal.

Techniques Used: Modification, SPR Assay

31) Product Images from "Valorization of Olive Pomace-Based Nutraceuticals as Antioxidants in Chemical, Food, and Biological Models"

Article Title: Valorization of Olive Pomace-Based Nutraceuticals as Antioxidants in Chemical, Food, and Biological Models

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

doi: 10.3390/molecules23082070

Inhibition of plasmid DNA strand scission and antioxidant activity of DOPE in liposome membrane model. ( a ): supercoiled (native) form of plasmid pBR322 DNA in electrophoresis gel after incubation with AAPH with or without the presence of antioxidants; ( b ): antioxidant activity against antioxidant activity against plasmid DNA strand scission expressed as IC50; ( c ): antioxidant activity of DOPE in liposome membrane model system. nDOPE-native dry olive pomace extract; hpDOPE-extract prepared with hydroxypropyl-β-cyclodextrin; ramDOPE-extract prepared with randomly methylated β-cyclodextrin; BHA: butylhydroxyanisol; IC 50 : half maximal inhibitory concentration; TBARS: thiobarbituric acid reactive substances. Different letters above data bars indicate significant difference ( p ≤ 0.05).
Figure Legend Snippet: Inhibition of plasmid DNA strand scission and antioxidant activity of DOPE in liposome membrane model. ( a ): supercoiled (native) form of plasmid pBR322 DNA in electrophoresis gel after incubation with AAPH with or without the presence of antioxidants; ( b ): antioxidant activity against antioxidant activity against plasmid DNA strand scission expressed as IC50; ( c ): antioxidant activity of DOPE in liposome membrane model system. nDOPE-native dry olive pomace extract; hpDOPE-extract prepared with hydroxypropyl-β-cyclodextrin; ramDOPE-extract prepared with randomly methylated β-cyclodextrin; BHA: butylhydroxyanisol; IC 50 : half maximal inhibitory concentration; TBARS: thiobarbituric acid reactive substances. Different letters above data bars indicate significant difference ( p ≤ 0.05).

Techniques Used: Inhibition, Plasmid Preparation, Antioxidant Activity Assay, Electrophoresis, Incubation, Methylation, Concentration Assay

32) Product Images from "Distinct molecular and cellular contributions to stabilizing selectin-mediated rolling under flow"

Article Title: Distinct molecular and cellular contributions to stabilizing selectin-mediated rolling under flow

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200204041

Effects of cellular perturbations on detachment resistance and rolling properties of sPSGL-1–coupled K562 cells on P-selectin. Cells were fixed with paraformaldehyde or treated with MβCD or cytochalasin D before coupling of sPSGL-1. Untreated and treated cells displayed equivalent surface densities of sPSGL-1 (unpublished data). (A and B) Detachment resistance and mean rolling velocities of nonfixed or fixed cells were measured as in Fig. 3 . (C) Frame-by-frame velocities of representative nonfixed and fixed cells rolling on P-selectin at 145 sites/μm 2 . (D) Mean velocities and variances of velocities for populations of nonfixed or fixed cells rolling on P-selectin at 145 sites/μm 2 . (E and F) sPSGL-1 was coupled to approximately sevenfold higher density on untreated cells, fixed cells, or cells treated with MβCD or cytochalasin D. Detachment resistance and mean rolling velocities were measured as in Fig. 3 . Cells treated with αCD, an inactive analogue of MβCD, or with DMSO, the diluent for cytochalasin D, rolled identically to untreated cells (unpublished data). The data represent the mean ± SD of five experiments.
Figure Legend Snippet: Effects of cellular perturbations on detachment resistance and rolling properties of sPSGL-1–coupled K562 cells on P-selectin. Cells were fixed with paraformaldehyde or treated with MβCD or cytochalasin D before coupling of sPSGL-1. Untreated and treated cells displayed equivalent surface densities of sPSGL-1 (unpublished data). (A and B) Detachment resistance and mean rolling velocities of nonfixed or fixed cells were measured as in Fig. 3 . (C) Frame-by-frame velocities of representative nonfixed and fixed cells rolling on P-selectin at 145 sites/μm 2 . (D) Mean velocities and variances of velocities for populations of nonfixed or fixed cells rolling on P-selectin at 145 sites/μm 2 . (E and F) sPSGL-1 was coupled to approximately sevenfold higher density on untreated cells, fixed cells, or cells treated with MβCD or cytochalasin D. Detachment resistance and mean rolling velocities were measured as in Fig. 3 . Cells treated with αCD, an inactive analogue of MβCD, or with DMSO, the diluent for cytochalasin D, rolled identically to untreated cells (unpublished data). The data represent the mean ± SD of five experiments.

Techniques Used:

33) Product Images from "A role for myosin-1A in the localization of a brush border disaccharidase"

Article Title: A role for myosin-1A in the localization of a brush border disaccharidase

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200310031

Effect of M β CD on DRMs isolated from BBE cells. (A) Density (dashed line, closed circle) and cholesterol profiles of Optiprep TM density gradients used to characterize DRMs from BBE cells with (solid line, filled square) or without (solid line, open square) MβCD treatment. (B) Immunoblots of gradient fractions (loaded for equal volume) demonstrate that cholesterol depletion is linked to a significant loss of SI and M1A from the lowest density fraction (asterisks). (C) Immunoblots of pellet and supernatant fractions from ConA pulldown experiments show that SI and M1A sediment with ConA-coated (ConA), but not uncoated (−Cntrl) agarose beads. (D) Immunoblots reveal that exposure of the ConA pellet (from C) to elevated temperature (37°C) or MβCD (see Materials and methods) results only in minor solubilization of M1A.
Figure Legend Snippet: Effect of M β CD on DRMs isolated from BBE cells. (A) Density (dashed line, closed circle) and cholesterol profiles of Optiprep TM density gradients used to characterize DRMs from BBE cells with (solid line, filled square) or without (solid line, open square) MβCD treatment. (B) Immunoblots of gradient fractions (loaded for equal volume) demonstrate that cholesterol depletion is linked to a significant loss of SI and M1A from the lowest density fraction (asterisks). (C) Immunoblots of pellet and supernatant fractions from ConA pulldown experiments show that SI and M1A sediment with ConA-coated (ConA), but not uncoated (−Cntrl) agarose beads. (D) Immunoblots reveal that exposure of the ConA pellet (from C) to elevated temperature (37°C) or MβCD (see Materials and methods) results only in minor solubilization of M1A.

Techniques Used: Isolation, Western Blot

34) Product Images from "Opa Proteins of Pathogenic Neisseriae Initiate Src Kinase-Dependent or Lipid Raft-Mediated Uptake via Distinct Human Carcinoembryonic Antigen-Related Cell Adhesion Molecule Isoforms ▿Opa Proteins of Pathogenic Neisseriae Initiate Src Kinase-Dependent or Lipid Raft-Mediated Uptake via Distinct Human Carcinoembryonic Antigen-Related Cell Adhesion Molecule Isoforms ▿ †"

Article Title: Opa Proteins of Pathogenic Neisseriae Initiate Src Kinase-Dependent or Lipid Raft-Mediated Uptake via Distinct Human Carcinoembryonic Antigen-Related Cell Adhesion Molecule Isoforms ▿Opa Proteins of Pathogenic Neisseriae Initiate Src Kinase-Dependent or Lipid Raft-Mediated Uptake via Distinct Human Carcinoembryonic Antigen-Related Cell Adhesion Molecule Isoforms ▿ †

Journal: Infection and Immunity

doi: 10.1128/IAI.01835-06

Disruption of membrane microdomains inhibits uptake by epithelial CEACAMs. (A and B) 293T cells were transfected with the empty control vector (pcDNA) or cDNA encoding CEACAM3 or CEACAM6. Transfected cells were infected for 60 min with Opa CEA -expressing N. gonorrhoeae (MOI, 20) and applied in gentamicin assays. Prior to infection, cells were pretreated or not with the indicated amounts of nystatin (A) or filipin (B). The bars represent mean values ± standard deviations from three independent experiments done in triplicate. Values are expressed as percentages of the untreated control value. (C) 293T cells expressing the indicated CEACAMs were infected for 60 min with Opa CEA -expressing gonococci and analyzed in gentamicin assays in the presence or absence of 20 μg/ml nystatin. The bars represent mean values ± standard deviations from three independent experiments done in triplicate. Values are expressed as percentages of the untreated control value.
Figure Legend Snippet: Disruption of membrane microdomains inhibits uptake by epithelial CEACAMs. (A and B) 293T cells were transfected with the empty control vector (pcDNA) or cDNA encoding CEACAM3 or CEACAM6. Transfected cells were infected for 60 min with Opa CEA -expressing N. gonorrhoeae (MOI, 20) and applied in gentamicin assays. Prior to infection, cells were pretreated or not with the indicated amounts of nystatin (A) or filipin (B). The bars represent mean values ± standard deviations from three independent experiments done in triplicate. Values are expressed as percentages of the untreated control value. (C) 293T cells expressing the indicated CEACAMs were infected for 60 min with Opa CEA -expressing gonococci and analyzed in gentamicin assays in the presence or absence of 20 μg/ml nystatin. The bars represent mean values ± standard deviations from three independent experiments done in triplicate. Values are expressed as percentages of the untreated control value.

Techniques Used: Transfection, Plasmid Preparation, Infection, Expressing

35) Product Images from "Binding of Human Immunodeficiency Virus Type 1 to Immature Dendritic Cells Can Occur Independently of DC-SIGN and Mannose Binding C-Type Lectin Receptors via a Cholesterol-Dependent Pathway"

Article Title: Binding of Human Immunodeficiency Virus Type 1 to Immature Dendritic Cells Can Occur Independently of DC-SIGN and Mannose Binding C-Type Lectin Receptors via a Cholesterol-Dependent Pathway

Journal: Journal of Virology

doi: 10.1128/JVI.77.23.12865-12874.2003

Binding of HIV-1 to DC is cholesterol dependent. DC were exposed to the endocytosis inhibitors and then incubated with FITC-labeled transferrin at 4°C. Cells were also stained with DAPI to help in the visualization of the cellular architecture. The images are the composite of a series of Z sections collected through the entire thickness of the cell monolayer and projected onto a two-dimensional plane. (A) At 4°C, transferrin-FITC can bind to its receptor but is not internalized, as evident from the staining observed predominantly at the plasma membrane. (B) Cells were shifted to 37°C for a period of 15 min and then monitored again for transferrin localization. Note the prominent intracellular green fluorescence indicative of uptake of transferrin-FITC. (C) Treatment with 500 μM amantadine prior to incubation with transferrin-FITC for 1 h at 37°C resulted in transferrin-FITC staining at the plasma membrane (indicated by white arrowheads) and completely abolished the intracellular staining, while treatment with 5 mM MβCD (D) had no effect on the intracellular accumulation of transferrin. (E) DC (4 × 10 4 cells) treated with the endocytosis inhibitors were exposed to Lai virus particles (30 ng of p24 gag ) for 2 h at 37°C. The mean level of virus (Lai) binding in the presence of endocytosis inhibitors was normalized to the level of virus binding in the absence of inhibitors (set at 100%). This experiment was performed at least five independent times with DC derived from five independent donors, and each experiment was performed in triplicate. The results shown are the averages of the mean percent binding (± standard deviations) observed for all the experiments. (F) DC (4 × 10 4 cells) were treated with amantadine (500 μM) and then exposed to Lai virus particles (30 ng of p24 gag ) for 2 h either at 4 or 37°C. (G) DC (4 × 10 4 cells) were treated with MβCD (5 mM) and then exposed to Lai virus particles (30 ng of p24 gag ) for 2 h either at 4 or 37°C. The percent virus (p24 gag ) binding values shown in panels F and G represent the means ± standard deviations of triplicate cultures. The results from one representative experiment out of three with DC derived from three independent donors are shown in panels F and G.
Figure Legend Snippet: Binding of HIV-1 to DC is cholesterol dependent. DC were exposed to the endocytosis inhibitors and then incubated with FITC-labeled transferrin at 4°C. Cells were also stained with DAPI to help in the visualization of the cellular architecture. The images are the composite of a series of Z sections collected through the entire thickness of the cell monolayer and projected onto a two-dimensional plane. (A) At 4°C, transferrin-FITC can bind to its receptor but is not internalized, as evident from the staining observed predominantly at the plasma membrane. (B) Cells were shifted to 37°C for a period of 15 min and then monitored again for transferrin localization. Note the prominent intracellular green fluorescence indicative of uptake of transferrin-FITC. (C) Treatment with 500 μM amantadine prior to incubation with transferrin-FITC for 1 h at 37°C resulted in transferrin-FITC staining at the plasma membrane (indicated by white arrowheads) and completely abolished the intracellular staining, while treatment with 5 mM MβCD (D) had no effect on the intracellular accumulation of transferrin. (E) DC (4 × 10 4 cells) treated with the endocytosis inhibitors were exposed to Lai virus particles (30 ng of p24 gag ) for 2 h at 37°C. The mean level of virus (Lai) binding in the presence of endocytosis inhibitors was normalized to the level of virus binding in the absence of inhibitors (set at 100%). This experiment was performed at least five independent times with DC derived from five independent donors, and each experiment was performed in triplicate. The results shown are the averages of the mean percent binding (± standard deviations) observed for all the experiments. (F) DC (4 × 10 4 cells) were treated with amantadine (500 μM) and then exposed to Lai virus particles (30 ng of p24 gag ) for 2 h either at 4 or 37°C. (G) DC (4 × 10 4 cells) were treated with MβCD (5 mM) and then exposed to Lai virus particles (30 ng of p24 gag ) for 2 h either at 4 or 37°C. The percent virus (p24 gag ) binding values shown in panels F and G represent the means ± standard deviations of triplicate cultures. The results from one representative experiment out of three with DC derived from three independent donors are shown in panels F and G.

Techniques Used: Binding Assay, Incubation, Labeling, Staining, Fluorescence, Derivative Assay

36) Product Images from "Exosomal miR-9 released from HIV Tat stimulated astrocytes mediates microglial migration"

Article Title: Exosomal miR-9 released from HIV Tat stimulated astrocytes mediates microglial migration

Journal: Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology

doi: 10.1007/s11481-018-9779-4

HIV Tat upregulates miR-9 in EVs released from astrocytes EVs isolated from A172 astrocyte cultures were characterized by Transmission Electron microscopy (A) . Scale bar 100 nm. (B) Western blot characterization of astrocyte EVs with exosome marker antibody against Tsg101, Alix, and CD63. (C) Size and particle distribution plots of isolated EVs from control A172 cells or A172 cells exposed to HIV Tat by NanoSight Tracking Analysis (NTA). The image shows increased size and number of vesicles released from A172 cells exposed to HIV Tat. (D) The time course of miR9 expresion in EVs released from human primary astrocytes exposed to HIV tat for 24 h, 48 h and 72 h. (E) A172 cells were pre-treated with exosome release inhibitor (DMA and MβCD) for 1 h, followed by Tat exposure for 24 h. Total RNA from EVs was analyzed by real-time PCR for miR-9. (F) Uptaken of EVs-TSG101-mcherry from A172 cells transfected with either pEF6.mCherry-TSG101 (left) or pcDNA3.1 (right) plasmid by BV-2 cells; TSG101 (Exosome marker, red), Iba-1 (microglia marker, green), DAPI (nucleus, blue). Scale bar = 20 µm. (G) BV-2 cells exposed to EVs derived from Tat stimulated A172 cells showed increased miR-9 compared with BV-2 cells exposed to control EVs or BV-2 cells not exposed to EVs. Data are presented as mean ± SD of four individual experiments. * P
Figure Legend Snippet: HIV Tat upregulates miR-9 in EVs released from astrocytes EVs isolated from A172 astrocyte cultures were characterized by Transmission Electron microscopy (A) . Scale bar 100 nm. (B) Western blot characterization of astrocyte EVs with exosome marker antibody against Tsg101, Alix, and CD63. (C) Size and particle distribution plots of isolated EVs from control A172 cells or A172 cells exposed to HIV Tat by NanoSight Tracking Analysis (NTA). The image shows increased size and number of vesicles released from A172 cells exposed to HIV Tat. (D) The time course of miR9 expresion in EVs released from human primary astrocytes exposed to HIV tat for 24 h, 48 h and 72 h. (E) A172 cells were pre-treated with exosome release inhibitor (DMA and MβCD) for 1 h, followed by Tat exposure for 24 h. Total RNA from EVs was analyzed by real-time PCR for miR-9. (F) Uptaken of EVs-TSG101-mcherry from A172 cells transfected with either pEF6.mCherry-TSG101 (left) or pcDNA3.1 (right) plasmid by BV-2 cells; TSG101 (Exosome marker, red), Iba-1 (microglia marker, green), DAPI (nucleus, blue). Scale bar = 20 µm. (G) BV-2 cells exposed to EVs derived from Tat stimulated A172 cells showed increased miR-9 compared with BV-2 cells exposed to control EVs or BV-2 cells not exposed to EVs. Data are presented as mean ± SD of four individual experiments. * P

Techniques Used: Isolation, Transmission Assay, Electron Microscopy, Western Blot, Marker, Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, Derivative Assay

37) Product Images from "The Dynamin Chemical Inhibitor Dynasore Impairs Cholesterol Trafficking and Sterol-Sensitive Genes Transcription in Human HeLa Cells and Macrophages"

Article Title: The Dynamin Chemical Inhibitor Dynasore Impairs Cholesterol Trafficking and Sterol-Sensitive Genes Transcription in Human HeLa Cells and Macrophages

Journal: PLoS ONE

doi: 10.1371/journal.pone.0029042

Effects of U18666A on the intracellular distribution of FC and LDL in HeLa cells and HMDM. HeLa cells and HMDM were respectively incubated for 6 h with 200 µg/mL LDL (A) or 50 µg/ml AcLDL (C) with 3 µg/ml U18666A or without (control) and stained with filipin to detect FC. (B–D) HeLa cells and HMDM were respectively incubated for 6 h with 200 µg/ml DiI-LDL (B) or 50 µg/ml DiI-AcLDL (D) with 3 µg/ml U18666A or without (control) and processed to visualize LDL distribution. Images were obtained using wide-field epifluorescence microscopy.
Figure Legend Snippet: Effects of U18666A on the intracellular distribution of FC and LDL in HeLa cells and HMDM. HeLa cells and HMDM were respectively incubated for 6 h with 200 µg/mL LDL (A) or 50 µg/ml AcLDL (C) with 3 µg/ml U18666A or without (control) and stained with filipin to detect FC. (B–D) HeLa cells and HMDM were respectively incubated for 6 h with 200 µg/ml DiI-LDL (B) or 50 µg/ml DiI-AcLDL (D) with 3 µg/ml U18666A or without (control) and processed to visualize LDL distribution. Images were obtained using wide-field epifluorescence microscopy.

Techniques Used: Incubation, Staining, Epifluorescence Microscopy

Effect of dynasore on LDL uptake and total cholesterol in HeLa cells and HMDM. Cells were incubated for 4 h with 0–200 µg/ml DiI-LDL (A) or 0–100 µg/ml DiI-AcLDL (C) at 37°C with 0.4% v/v DMSO (control) or 80 µM dynasore. The total amount of endocytosed DiI-LDL or DiI-AcLDL was measured by flow cytometry. Values represent the mean ± SD of triplicate experiments. Total cholesterol was quantified in HeLa cells (B) and HMDM (D) after 4 h of LDL uptake with 0.4% v/v DMSO (control) or 80 µM dynasore. Each value is the mean ± SD of triplicate experiments and expressed as nanomoles per mg of cell proteins.
Figure Legend Snippet: Effect of dynasore on LDL uptake and total cholesterol in HeLa cells and HMDM. Cells were incubated for 4 h with 0–200 µg/ml DiI-LDL (A) or 0–100 µg/ml DiI-AcLDL (C) at 37°C with 0.4% v/v DMSO (control) or 80 µM dynasore. The total amount of endocytosed DiI-LDL or DiI-AcLDL was measured by flow cytometry. Values represent the mean ± SD of triplicate experiments. Total cholesterol was quantified in HeLa cells (B) and HMDM (D) after 4 h of LDL uptake with 0.4% v/v DMSO (control) or 80 µM dynasore. Each value is the mean ± SD of triplicate experiments and expressed as nanomoles per mg of cell proteins.

Techniques Used: Incubation, Flow Cytometry, Cytometry

Effects of dynasore on the intracellular distribution of FC and LDL in HeLa cells and HMDM. (A) Hela cells were loaded with 200 µg/ml LDL for 24 h. Cells were then treated for 6 h with 80 µM dynasore or without (control) and stained with filipin to detect FC. (B) Cells were treated as described above with 200 µg/ml DiI-LDL. (C) HMDM were incubated for 6 h in LPDS medium containing 50 µg/ml DiI-AcLDL with 80 µM dynasore or without (control). (D) HMDM were loaded with 50 µg/ml DiI-AcLDL for 24 h and then treated for 6 h with 80 µM dynasore or without (control). Images were obtained using wide-field epifluorescence microscopy. Scale bars, 10 µm.
Figure Legend Snippet: Effects of dynasore on the intracellular distribution of FC and LDL in HeLa cells and HMDM. (A) Hela cells were loaded with 200 µg/ml LDL for 24 h. Cells were then treated for 6 h with 80 µM dynasore or without (control) and stained with filipin to detect FC. (B) Cells were treated as described above with 200 µg/ml DiI-LDL. (C) HMDM were incubated for 6 h in LPDS medium containing 50 µg/ml DiI-AcLDL with 80 µM dynasore or without (control). (D) HMDM were loaded with 50 µg/ml DiI-AcLDL for 24 h and then treated for 6 h with 80 µM dynasore or without (control). Images were obtained using wide-field epifluorescence microscopy. Scale bars, 10 µm.

Techniques Used: Staining, Incubation, Epifluorescence Microscopy

U18666A impairs cellular cholesterol efflux from HMDM. Cells were incubated with 100 µg/ml AcLDL for 6 h and treated with 3 µg/ml U18666A or without (control). The cellular cholesterol efflux to 10 µg/ml apoA-I or 15 µg/ml HDL-PL before (A) and after (B) stimulation of ABCA1 and ABCG1 expression by the LXR/RXR agonists was quantified. Results are expressed as the percentage of the quantity of released cellular cholesterol into the medium to the total quantity of cholesterol in cells and medium. Each value is the mean of triplicate experiments. (C) Relative quantification of ABCA1 and ABCG1 transporter genes levels expressed as fold-variation over control (DMSO/LPDS) after normalization. All CT determinations were made in triplicate. (D) Passive cholesterol efflux to 1 mg/ml MâCD was quantified as above.
Figure Legend Snippet: U18666A impairs cellular cholesterol efflux from HMDM. Cells were incubated with 100 µg/ml AcLDL for 6 h and treated with 3 µg/ml U18666A or without (control). The cellular cholesterol efflux to 10 µg/ml apoA-I or 15 µg/ml HDL-PL before (A) and after (B) stimulation of ABCA1 and ABCG1 expression by the LXR/RXR agonists was quantified. Results are expressed as the percentage of the quantity of released cellular cholesterol into the medium to the total quantity of cholesterol in cells and medium. Each value is the mean of triplicate experiments. (C) Relative quantification of ABCA1 and ABCG1 transporter genes levels expressed as fold-variation over control (DMSO/LPDS) after normalization. All CT determinations were made in triplicate. (D) Passive cholesterol efflux to 1 mg/ml MâCD was quantified as above.

Techniques Used: Incubation, Expressing

Dynasore impairs cellular cholesterol efflux from HMDM. HMDM were incubated for 6 h with 100 µg/ml AcLDL with 80 µM dynasore or without (control). The cellular cholesterol efflux to 10 µg/ml apoA-I or 15 µg/ml HDL-PL before (A) and after (B) stimulation of ABCA1 and ABCG1 expression by the LXR/RXR agonists was quantified. Results are expressed as the percentage of the quantity of released cellular cholesterol into the medium to the total quantity of cholesterol in cells and medium. Each value is the mean of triplicate experiments. (C) Relative quantification of ABCA1 and ABCG1 transporter genes levels was expressed as fold-variation over control (DMSO/LPDS) after normalization. All CT determinations were made in triplicate. (D) Passive cholesterol efflux to 1 mg/ml MâCD was quantified as above.
Figure Legend Snippet: Dynasore impairs cellular cholesterol efflux from HMDM. HMDM were incubated for 6 h with 100 µg/ml AcLDL with 80 µM dynasore or without (control). The cellular cholesterol efflux to 10 µg/ml apoA-I or 15 µg/ml HDL-PL before (A) and after (B) stimulation of ABCA1 and ABCG1 expression by the LXR/RXR agonists was quantified. Results are expressed as the percentage of the quantity of released cellular cholesterol into the medium to the total quantity of cholesterol in cells and medium. Each value is the mean of triplicate experiments. (C) Relative quantification of ABCA1 and ABCG1 transporter genes levels was expressed as fold-variation over control (DMSO/LPDS) after normalization. All CT determinations were made in triplicate. (D) Passive cholesterol efflux to 1 mg/ml MâCD was quantified as above.

Techniques Used: Incubation, Expressing

U18666A inhibits ACAT activity and sterol-sensitive genes regulation in HeLa cells and HMDM. Cells were grown in LPDS medium for 48 h and further incubated for 6 h with 200 µg/ml LDL (A) or 50 µg/ml AcLDL (B) with 3 µg/ml U18666A or without (control). Relative quantification of LDLR, HMGCoAR, and SREBF-2 genes in HeLa cells (A) or HMDM (B) was expressed as fold-variation over control (LPDS/DMSO) after normalization. All CT determinations were made in triplicate. The total amount of CE was quantified HeLa cells (C) and in HMDM (D) and expressed as the percent of the total amount of cholesterol. ACAT-dependent ester formation was measured with 10 µg/ml ACAT inhibitor (grey bars). Cholesteryl myristate formation was measured in HeLa cells (E) or HMDM (F) with 3 µg/ml U18666A or without (control). Cholesteryl myristate was expressed in nmol/mg protein. Each value is the mean of triplicate experiments.
Figure Legend Snippet: U18666A inhibits ACAT activity and sterol-sensitive genes regulation in HeLa cells and HMDM. Cells were grown in LPDS medium for 48 h and further incubated for 6 h with 200 µg/ml LDL (A) or 50 µg/ml AcLDL (B) with 3 µg/ml U18666A or without (control). Relative quantification of LDLR, HMGCoAR, and SREBF-2 genes in HeLa cells (A) or HMDM (B) was expressed as fold-variation over control (LPDS/DMSO) after normalization. All CT determinations were made in triplicate. The total amount of CE was quantified HeLa cells (C) and in HMDM (D) and expressed as the percent of the total amount of cholesterol. ACAT-dependent ester formation was measured with 10 µg/ml ACAT inhibitor (grey bars). Cholesteryl myristate formation was measured in HeLa cells (E) or HMDM (F) with 3 µg/ml U18666A or without (control). Cholesteryl myristate was expressed in nmol/mg protein. Each value is the mean of triplicate experiments.

Techniques Used: Activity Assay, Incubation

Dynasore decreases the production of cholesterol esters from LDL- or AcLDL-derived cholesterol. HeLa cells or HMDM were respectively incubated with 200 µg/ml LDL (A) or 50 µg/ml AcLDL (C) and treated for 6 h with 80 µM dynasore or without (control). The total amount of CE was quantified and expressed as the percent of the total amount of cholesterol. ACAT-dependent ester formation was measured with 10 µg/ml ACAT inhibitor (grey bars). The production of cholesteryl myristate was measured in HeLa cells (B) or HMDM (D) treated or not (control) with 80 µM dynasore. Cholesteryl myristate was expressed in nmol/mg protein. Each value is the mean of triplicate experiments.
Figure Legend Snippet: Dynasore decreases the production of cholesterol esters from LDL- or AcLDL-derived cholesterol. HeLa cells or HMDM were respectively incubated with 200 µg/ml LDL (A) or 50 µg/ml AcLDL (C) and treated for 6 h with 80 µM dynasore or without (control). The total amount of CE was quantified and expressed as the percent of the total amount of cholesterol. ACAT-dependent ester formation was measured with 10 µg/ml ACAT inhibitor (grey bars). The production of cholesteryl myristate was measured in HeLa cells (B) or HMDM (D) treated or not (control) with 80 µM dynasore. Cholesteryl myristate was expressed in nmol/mg protein. Each value is the mean of triplicate experiments.

Techniques Used: Derivative Assay, Incubation

Effect of U18666A on LDL uptake and total cholesterol in HeLa cells and HMDM. LDL uptake was measured in HeLa cells (A) and HMDM (C) after incubation at 37°C for 4 h with 0–200 µg/ml DiI-LDL or 0–100 µg/ml DiI-AcLDL, respectively, with 3 µg/ml U18666A or without (control). The amount of endocytosed DiI-LDL and DiI-AcLDL was measured by flow cytometry. Values represent the mean ± SD of triplicate experiments. Total cholesterol was quantified in HeLa cells (B) and HMDM (D) after 4 h of LDL uptake with 3 µg/ml U18666A or without (control). Each value is the mean ± SD of triplicate experiments and expressed as nanomoles per mg of cell proteins.
Figure Legend Snippet: Effect of U18666A on LDL uptake and total cholesterol in HeLa cells and HMDM. LDL uptake was measured in HeLa cells (A) and HMDM (C) after incubation at 37°C for 4 h with 0–200 µg/ml DiI-LDL or 0–100 µg/ml DiI-AcLDL, respectively, with 3 µg/ml U18666A or without (control). The amount of endocytosed DiI-LDL and DiI-AcLDL was measured by flow cytometry. Values represent the mean ± SD of triplicate experiments. Total cholesterol was quantified in HeLa cells (B) and HMDM (D) after 4 h of LDL uptake with 3 µg/ml U18666A or without (control). Each value is the mean ± SD of triplicate experiments and expressed as nanomoles per mg of cell proteins.

Techniques Used: Incubation, Flow Cytometry, Cytometry

Dynasore blocks sterol-sensitive genes regulation in HeLa cells and HMDM. (A) Kinetics of LDLR expression analyzed by RT-PCR. HeLa cells were grown in LPDS medium for 48 h and further incubated for the indicated times with medium containing either LPDS, 200 µg/ml LDL, or 200 µg/ml LDL with 80 µM dynasore. (B) The expression level of sterol-sensitive genes (LDLR, HMGCoAR and SREBF-2) was quantified after 6 h in HeLa cells grown in LPDS, with 200 µg/ml LDL or 200 µg/ml LDL with 80 µM dynasore, as indicated. (C) The same experiment was performed in HMDM with 50 µg/ml AcLDL. Relative quantification of LDLR, HMGCoAR, and SREBF-2 genes in HeLa cells or HMDM was expressed as fold-variation over control (LPDS/DMSO) after normalization. All CT determinations were made in triplicate.
Figure Legend Snippet: Dynasore blocks sterol-sensitive genes regulation in HeLa cells and HMDM. (A) Kinetics of LDLR expression analyzed by RT-PCR. HeLa cells were grown in LPDS medium for 48 h and further incubated for the indicated times with medium containing either LPDS, 200 µg/ml LDL, or 200 µg/ml LDL with 80 µM dynasore. (B) The expression level of sterol-sensitive genes (LDLR, HMGCoAR and SREBF-2) was quantified after 6 h in HeLa cells grown in LPDS, with 200 µg/ml LDL or 200 µg/ml LDL with 80 µM dynasore, as indicated. (C) The same experiment was performed in HMDM with 50 µg/ml AcLDL. Relative quantification of LDLR, HMGCoAR, and SREBF-2 genes in HeLa cells or HMDM was expressed as fold-variation over control (LPDS/DMSO) after normalization. All CT determinations were made in triplicate.

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Incubation

38) Product Images from "The LTB4–BLT1 axis regulates the polarized trafficking of chemoattractant GPCRs during neutrophil chemotaxis"

Article Title: The LTB4–BLT1 axis regulates the polarized trafficking of chemoattractant GPCRs during neutrophil chemotaxis

Journal: Journal of Cell Science

doi: 10.1242/jcs.217422

FPR1 undergoes CDC42-PAK1-actin-dependent endocytosis. (A) A representative confocal image of PMNs treated with fNLFNYK–FITC (2 nM; green) for 10 min, fixed and stained with phalloidin (red). Turquoise arrowheads highlight the fNLFNYK vesicles enriched with F-actin. Scale bars: 5 µm. Images are representative of at least three independent experiments. (B) Graph depicting the extent of internalization of fNLFNYK–FITC in PMNs pretreated with vehicle [0.2% (v/v) DMSO] or inhibitors or activators (denoted with an ‘i’ and ‘a’ after the protein targeted, respectively) (25 µM nocadazole; 100 nM Lat A; 100 µM CK666; 100 µM CK689; 5 µM IPA3; 25 µM EHT1864; 100 µM ML141; 100 µM ZCL278; 50 µM EIPA; 20 µM Brefeldin A), stimulated with fNLFNYK–FITC (2 nM) for 10 min and fixed. The extent of internalization of fNLFNYK was quantified and represented as the percentage of vehicle control from n =4 independent experiments. Findings are represented as mean±s.e.m. **** P ≤0.0001 (one-way ANOVA with Dunnett's multiple comparisons test). (C) Representative confocal images of Scr , CHC −/− , Cav1 −/− , FLT2 −/− , PAK1 −/− and CDC42 −/− dPLBs treated with fNLFNYK–FITC (10 nM; green) for 15 min, fixed and stained with phalloidin (red) and DAPI (blue). Scale bars: 10 µm. Images are representative of three independent experiments. (D) Graph depicting the extent of internalization of fNLFNYK–FITC in Scr , CHC −/− , Cav1 −/− , FLT2 −/− , PAK1 −/− and CDC42 −/− dPLBs treated with fNLFNYK–FITC (10 nM) for 15 min and fixed. The extent of internalization of fNLFNYK was quantified and represented as a percentage of Scr control from n =4 independent experiments. Findings are represented as mean±s.e.m. *** P ≤0.001 (one-way ANOVA with Dunnett's multiple comparisons test).
Figure Legend Snippet: FPR1 undergoes CDC42-PAK1-actin-dependent endocytosis. (A) A representative confocal image of PMNs treated with fNLFNYK–FITC (2 nM; green) for 10 min, fixed and stained with phalloidin (red). Turquoise arrowheads highlight the fNLFNYK vesicles enriched with F-actin. Scale bars: 5 µm. Images are representative of at least three independent experiments. (B) Graph depicting the extent of internalization of fNLFNYK–FITC in PMNs pretreated with vehicle [0.2% (v/v) DMSO] or inhibitors or activators (denoted with an ‘i’ and ‘a’ after the protein targeted, respectively) (25 µM nocadazole; 100 nM Lat A; 100 µM CK666; 100 µM CK689; 5 µM IPA3; 25 µM EHT1864; 100 µM ML141; 100 µM ZCL278; 50 µM EIPA; 20 µM Brefeldin A), stimulated with fNLFNYK–FITC (2 nM) for 10 min and fixed. The extent of internalization of fNLFNYK was quantified and represented as the percentage of vehicle control from n =4 independent experiments. Findings are represented as mean±s.e.m. **** P ≤0.0001 (one-way ANOVA with Dunnett's multiple comparisons test). (C) Representative confocal images of Scr , CHC −/− , Cav1 −/− , FLT2 −/− , PAK1 −/− and CDC42 −/− dPLBs treated with fNLFNYK–FITC (10 nM; green) for 15 min, fixed and stained with phalloidin (red) and DAPI (blue). Scale bars: 10 µm. Images are representative of three independent experiments. (D) Graph depicting the extent of internalization of fNLFNYK–FITC in Scr , CHC −/− , Cav1 −/− , FLT2 −/− , PAK1 −/− and CDC42 −/− dPLBs treated with fNLFNYK–FITC (10 nM) for 15 min and fixed. The extent of internalization of fNLFNYK was quantified and represented as a percentage of Scr control from n =4 independent experiments. Findings are represented as mean±s.e.m. *** P ≤0.001 (one-way ANOVA with Dunnett's multiple comparisons test).

Techniques Used: Staining

39) Product Images from "Peste des Petits Ruminants Virus Enters Caprine Endometrial Epithelial Cells via the Caveolae-Mediated Endocytosis Pathway"

Article Title: Peste des Petits Ruminants Virus Enters Caprine Endometrial Epithelial Cells via the Caveolae-Mediated Endocytosis Pathway

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2018.00210

CME is not the pathway for PPRV entry into EECs. (A–C) Effect of CPZ treatment on PPRV entry and infection. (A) CLSM analysis of clathrin (green), viral particles (red) and cell nuclei (blue) in PPRV-infected EECs pre-treated with CPZ. The effect of CPZ on Alexa Fluor 594–TF uptake was used as a positive control (red fluorescence). (B) Western blot analysis of the entry of PPRV into CPZ-treated cells. β-actin was used as an internal control. (C) Virus titration results of PPRV in CPZ -treated cells. (D) CLSM analysis of TRITC-phalloidin (red), anti-PPRV (green) and DAPI (blue) in PPRV-infected EECs pre-treated with CPZ. (E) Western blot analysis of the entry of PPRV into shRNA-transfected cells. (F) Efficiency of CHC downregulation was analyzed by immunoblotting. (G) The relative quantification of viral proteins was determined by densitometry against β-actin in shRNA-transfected cells. (H) Virus titration analysis of PPRV in shRNA-transfected cells. The bars indicate the mean ± SD from three independent experiments. SD, standard deviation; ∗ P
Figure Legend Snippet: CME is not the pathway for PPRV entry into EECs. (A–C) Effect of CPZ treatment on PPRV entry and infection. (A) CLSM analysis of clathrin (green), viral particles (red) and cell nuclei (blue) in PPRV-infected EECs pre-treated with CPZ. The effect of CPZ on Alexa Fluor 594–TF uptake was used as a positive control (red fluorescence). (B) Western blot analysis of the entry of PPRV into CPZ-treated cells. β-actin was used as an internal control. (C) Virus titration results of PPRV in CPZ -treated cells. (D) CLSM analysis of TRITC-phalloidin (red), anti-PPRV (green) and DAPI (blue) in PPRV-infected EECs pre-treated with CPZ. (E) Western blot analysis of the entry of PPRV into shRNA-transfected cells. (F) Efficiency of CHC downregulation was analyzed by immunoblotting. (G) The relative quantification of viral proteins was determined by densitometry against β-actin in shRNA-transfected cells. (H) Virus titration analysis of PPRV in shRNA-transfected cells. The bars indicate the mean ± SD from three independent experiments. SD, standard deviation; ∗ P

Techniques Used: Infection, Confocal Laser Scanning Microscopy, Positive Control, Fluorescence, Western Blot, Titration, shRNA, Transfection, Standard Deviation

40) Product Images from "Cholesterol Depletion Reduces Entry of Campylobacter jejuni Cytolethal Distending Toxin and Attenuates Intoxication of Host Cells ▿"

Article Title: Cholesterol Depletion Reduces Entry of Campylobacter jejuni Cytolethal Distending Toxin and Attenuates Intoxication of Host Cells ▿

Journal: Infection and Immunity

doi: 10.1128/IAI.05175-11

Depletion of cholesterol reduces CDT holotoxin binding to cells. CHO-K1 cells were left untreated (upper) or treated with 10 mM MβCD (lower) for 1 h at 37°C prior to incubation with CDT holotoxin (200 nM). After incubation for 2 h at 4°C, the cells were probed with control preimmune serum or individual antiserum against each CDT subunit and stained with FITC-conjugated anti-mouse IgG. The level of binding activity was analyzed by flow cytometry for FITC fluorescence. The results represent the means and standard deviations from three independent experiments. The lower right panel shows the quantitative data of the CDT binding activity. An asterisk indicates significance compared to results for each untreated MβCD group, as determined by Student's t test ( P
Figure Legend Snippet: Depletion of cholesterol reduces CDT holotoxin binding to cells. CHO-K1 cells were left untreated (upper) or treated with 10 mM MβCD (lower) for 1 h at 37°C prior to incubation with CDT holotoxin (200 nM). After incubation for 2 h at 4°C, the cells were probed with control preimmune serum or individual antiserum against each CDT subunit and stained with FITC-conjugated anti-mouse IgG. The level of binding activity was analyzed by flow cytometry for FITC fluorescence. The results represent the means and standard deviations from three independent experiments. The lower right panel shows the quantitative data of the CDT binding activity. An asterisk indicates significance compared to results for each untreated MβCD group, as determined by Student's t test ( P

Techniques Used: Binding Assay, Incubation, Staining, Activity Assay, Flow Cytometry, Cytometry, Fluorescence

Sufficient cellular cholesterol is essential for CdtA and CdtC binding to CHO-K1 cells. The cells were left untreated (upper) or treated with 10 mM MβCD (lower) for 1 h at 37°C, followed by exposure to 200 nM the individual recombinant C. jejuni CDT proteins. After incubation with the individual CDT proteins for 2 h at 4°C, the cells were stained with control preimmune serum or individual antiserum against each CDT subunit and stained with FITC-conjugated anti-mouse IgG. Binding activity was assessed by flow cytometry. The numbers represent the mean channel fluorescence (MCF). The quantitative data represent the means and standard deviations from three independent experiments and are shown in the lower right panel. Statistical analysis was calculated using Student's t test compared to each untreated MβCD group. An asterisk indicates statistical significance ( P
Figure Legend Snippet: Sufficient cellular cholesterol is essential for CdtA and CdtC binding to CHO-K1 cells. The cells were left untreated (upper) or treated with 10 mM MβCD (lower) for 1 h at 37°C, followed by exposure to 200 nM the individual recombinant C. jejuni CDT proteins. After incubation with the individual CDT proteins for 2 h at 4°C, the cells were stained with control preimmune serum or individual antiserum against each CDT subunit and stained with FITC-conjugated anti-mouse IgG. Binding activity was assessed by flow cytometry. The numbers represent the mean channel fluorescence (MCF). The quantitative data represent the means and standard deviations from three independent experiments and are shown in the lower right panel. Statistical analysis was calculated using Student's t test compared to each untreated MβCD group. An asterisk indicates statistical significance ( P

Techniques Used: Binding Assay, Recombinant, Incubation, Staining, Activity Assay, Flow Cytometry, Cytometry, Fluorescence

Depletion of cholesterol prevents the nuclear localization of C. jejuni CdtB. (A) CHO-K1 cells were untreated or treated with 10 mM MβCD for 1 h prior to exposure to 200 nM CDT holotoxin at 37°C for the indicated times. The cells were washed and probed with anti-CdtB antiserum, followed by staining with FITC-conjugated anti-mouse IgG. The stained cells then were analyzed by confocal microscopy. Scale bars, 10 μm. (B) The nuclear fraction from cell lysates was prepared from CHO-K1 cells untreated or treated with 10 mM MβCD for 1 h, followed by incubation at 37°C in the presence of CDT holotoxin for the indicated times. CdtB in the nucleus of cell lysates was detected by Western blotting. The results represent three independent experiments. PCNA was used as a loading control for the nuclear fraction of cell lysates. (C) Protein expression levels were analyzed using scanning densitometry. The lower right panel shows the quantitative data for the nuclear CdtB signal. An asterisk indicates P
Figure Legend Snippet: Depletion of cholesterol prevents the nuclear localization of C. jejuni CdtB. (A) CHO-K1 cells were untreated or treated with 10 mM MβCD for 1 h prior to exposure to 200 nM CDT holotoxin at 37°C for the indicated times. The cells were washed and probed with anti-CdtB antiserum, followed by staining with FITC-conjugated anti-mouse IgG. The stained cells then were analyzed by confocal microscopy. Scale bars, 10 μm. (B) The nuclear fraction from cell lysates was prepared from CHO-K1 cells untreated or treated with 10 mM MβCD for 1 h, followed by incubation at 37°C in the presence of CDT holotoxin for the indicated times. CdtB in the nucleus of cell lysates was detected by Western blotting. The results represent three independent experiments. PCNA was used as a loading control for the nuclear fraction of cell lysates. (C) Protein expression levels were analyzed using scanning densitometry. The lower right panel shows the quantitative data for the nuclear CdtB signal. An asterisk indicates P

Techniques Used: Staining, Confocal Microscopy, Incubation, Western Blot, Expressing

Sufficient cellular cholesterol is essential for C. jejuni CDT-induced cell cycle arrest. CHO-K1 cells were preexposed to medium alone (A, D, and E), 5 mM MβCD (B and F), 10 mM MβCD (C and G), or 10 mM MβCD and replenished with cholesterol (400 μg/ml) for 1 h at 37°C (H). The cells then were incubated for 48 h at 37°C in the presence of medium (A), ICRF-193 (D), and C. jejuni CDT holotoxin (E to H). The cell cycle distribution was based on the DNA content, which was analyzed by flow cytometry. The percentage of cells in the G 0 /G 1 , S, and G 2 /M phases of the cell cycle are indicated at the right of each histogram. (I) The percentage of cells in the G 2 /M phase were calculated and plotted as intensity histograms. The results represent three independent experiments. An asterisk indicates statistical significance ( P
Figure Legend Snippet: Sufficient cellular cholesterol is essential for C. jejuni CDT-induced cell cycle arrest. CHO-K1 cells were preexposed to medium alone (A, D, and E), 5 mM MβCD (B and F), 10 mM MβCD (C and G), or 10 mM MβCD and replenished with cholesterol (400 μg/ml) for 1 h at 37°C (H). The cells then were incubated for 48 h at 37°C in the presence of medium (A), ICRF-193 (D), and C. jejuni CDT holotoxin (E to H). The cell cycle distribution was based on the DNA content, which was analyzed by flow cytometry. The percentage of cells in the G 0 /G 1 , S, and G 2 /M phases of the cell cycle are indicated at the right of each histogram. (I) The percentage of cells in the G 2 /M phase were calculated and plotted as intensity histograms. The results represent three independent experiments. An asterisk indicates statistical significance ( P

Techniques Used: Incubation, Flow Cytometry, Cytometry

Cholesterol is important for CDT association and intoxication of cells. (A) Cells from the indicated lines were untreated or treated with 5 mM (for AGS cells) or 10 mM (for other cells) MβCD for 1 h at 37°C, followed by exposure to 200 nM CDT holotoxin for 48 h. Cell cycle distribution was analyzed using flow cytometry. (B) Cells from the indicated lines were untreated or treated with 5 mM (AGS cells) or 10 mM (other cells) MβCD for 1 h at 37°C, followed by incubation with the individual CDT proteins for 2 h at 4°C. The binding activity of each CDT protein was assessed by flow cytometry for FITC fluorescence. The results represent the means and standard deviations from three independent experiments. An asterisk indicates P
Figure Legend Snippet: Cholesterol is important for CDT association and intoxication of cells. (A) Cells from the indicated lines were untreated or treated with 5 mM (for AGS cells) or 10 mM (for other cells) MβCD for 1 h at 37°C, followed by exposure to 200 nM CDT holotoxin for 48 h. Cell cycle distribution was analyzed using flow cytometry. (B) Cells from the indicated lines were untreated or treated with 5 mM (AGS cells) or 10 mM (other cells) MβCD for 1 h at 37°C, followed by incubation with the individual CDT proteins for 2 h at 4°C. The binding activity of each CDT protein was assessed by flow cytometry for FITC fluorescence. The results represent the means and standard deviations from three independent experiments. An asterisk indicates P

Techniques Used: Flow Cytometry, Cytometry, Incubation, Binding Assay, Activity Assay, Fluorescence

Cholesterol depletion in CHO-K1 cells by treatment with MβCD. (A) CHO-K1 cells were treated with 10 mM MβCD at 37°C and incubated for the indicated times. The cells were harvested and subjected to cold detergent extraction using 1% Triton X-100, followed by centrifugation to isolate the DRM fraction. The prepared total cell lysates and DRM fraction then were analyzed for cholesterol concentration as described in Materials and Methods. (B) CHO-K1 cells were treated with various concentrations of MβCD (0, 2.5, 5, 10, and 20 mM) for 1 h. Whole-cell lysates and the DRM fraction then were prepared for cholesterol level analysis. (C) Cell viability was barely influenced after treatment with 0 to 20 mM MβCD, as determined by the trypan blue exclusion assay. The data represent the means and standard deviations from three independent experiments. An asterisk indicates P
Figure Legend Snippet: Cholesterol depletion in CHO-K1 cells by treatment with MβCD. (A) CHO-K1 cells were treated with 10 mM MβCD at 37°C and incubated for the indicated times. The cells were harvested and subjected to cold detergent extraction using 1% Triton X-100, followed by centrifugation to isolate the DRM fraction. The prepared total cell lysates and DRM fraction then were analyzed for cholesterol concentration as described in Materials and Methods. (B) CHO-K1 cells were treated with various concentrations of MβCD (0, 2.5, 5, 10, and 20 mM) for 1 h. Whole-cell lysates and the DRM fraction then were prepared for cholesterol level analysis. (C) Cell viability was barely influenced after treatment with 0 to 20 mM MβCD, as determined by the trypan blue exclusion assay. The data represent the means and standard deviations from three independent experiments. An asterisk indicates P

Techniques Used: Incubation, Centrifugation, Concentration Assay, Trypan Blue Exclusion Assay

CdtA and CdtC are enriched in detergent-resistant membrane (DRM) fractions. CHO-K1 cells were left untreated or treated with 10 mM MβCD for 1 h prior to incubation with C. jejuni CDT holotoxin (200 nM) for 2 h at 37°C. The cells then were subjected to cold detergent extraction using 1% Triton X-100, followed by centrifugation to separate the DRM and detergent-soluble (S) fractions. (A) Each fraction was subjected to Western blot analysis using antibodies against caveolin-1 and CD71 and individual antisera specific to CdtA, CdtB, and CdtC. The results are representative of one of three independent experiments. (B) The protein expression levels were analyzed using scanning densitometry. The protein expression levels represent the relative distribution (percent) of each protein within the DRM and soluble fractions. MβCD, methyl-β-cyclodextrin.
Figure Legend Snippet: CdtA and CdtC are enriched in detergent-resistant membrane (DRM) fractions. CHO-K1 cells were left untreated or treated with 10 mM MβCD for 1 h prior to incubation with C. jejuni CDT holotoxin (200 nM) for 2 h at 37°C. The cells then were subjected to cold detergent extraction using 1% Triton X-100, followed by centrifugation to separate the DRM and detergent-soluble (S) fractions. (A) Each fraction was subjected to Western blot analysis using antibodies against caveolin-1 and CD71 and individual antisera specific to CdtA, CdtB, and CdtC. The results are representative of one of three independent experiments. (B) The protein expression levels were analyzed using scanning densitometry. The protein expression levels represent the relative distribution (percent) of each protein within the DRM and soluble fractions. MβCD, methyl-β-cyclodextrin.

Techniques Used: Incubation, Centrifugation, Western Blot, Expressing

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Article Snippet: .. Briefly, the invasion assays were performed in 0.5 ml of DMEM containing 0.1% BSA (Sigma) for 3 h at 37°C, and the number of intracellular parasites was estimated on Giemsa-stained coverslips by microscopy. ..

SDS Page:

Article Title: In search of a function of Manduca sexta hemolymph protease-1 in the innate immune system
Article Snippet: .. To detect possible N -linked glycosylation, proHP1 (0.1 µg) was treated with 1×glycoprotein denaturing buffer (Sigma- Aldrich) at 100°C for 10 min. After adding 1/10 volume each of 10×G7 buffer (50 mM sodium phosphate, pH 7.5) and 10% Nonidet P-40, the protein was incubated with 2.5 µl PNGase F (Sigma-Aldrich) at 37°C for 1 h. To detect O -linked glycosylation, proHP1 (0.1 µg) was reacted with 2 µl O -glycosidase (Sigma) in 1×reaction buffer at 37°C for 3 h. The reaction mixtures, as well as untreated control, were separated by 10% SDS-PAGE, electrotransferred to nitrocellulose membrane, and detected with HP1a antibodies. .. ProHP1a and proHP1b were amplified from their respective full-length cDNAs by PCR using forward (j1351 5’- GAATTC GAGATTACTCCGAACTGCA-3’, Eco RI site underlined) and reverse (j587, 5’- CTCGAG CCGTGCATTCTCCATG-3’, Xho I site underlined) primers.

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  • 94
    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
    https://www.bioz.com/result/cytochrome c reduction/product/Millipore
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    85
    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
    https://www.bioz.com/result/meβcd/product/Millipore
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    91
    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
    https://www.bioz.com/result/mβcd powder/product/Millipore
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    mβcd powder - by Bioz Stars, 2020-09
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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