Structured Review

Millipore dynasore
Pharmacological and genetic inhibition of Dyn2 function also reduces starvation-induced LD breakdown. HuH-7 (A) and Hep3B (B) hepatocytes were loaded with 150 µM oleate overnight and starved for 48 h in medium containing 0.1% FBS in the presence of Dyn2 inhibitors or DMSO as indicated. Representative images of inhibitor-treated and control cells (stained with Oil Red O) are shown in A and B together with the quantitation of the average LD area per cell from three independent experiments. Pharmacological inhibitors used were: <t>Dynasore</t> (inhibits Dyn2 GTPase activity), MiTMAB (targets PH domain and interferes with membrane binding), Dynole 34-2 (allosteric GTPase inhibitor), and Dynole 31-2 (negative control for Dynole 34-2). Bars, 20 µM. (C) Representative images from control and Dyn2 knockout MEFs after an overnight lipid loading with 400 µM oleate for 17 h. Knockout of Dyn2 was induced by treatment with 2 µM 4-hydroxy-tamoxifen for 7 d and was confirmed by immunostaining of endogenous Dyn2 (top row) and by immunoblot (D). Bars, 20 µM. (E and F) Average LD number (E) and area (F) per cell from five independent experiments. All data are represented as mean ± SE. *, P ≤ 0.05; **, P ≤ 0.01. (G) Whole-cell lysates and LD fractions isolated from HuH-7 hepatocytes under resting or starved (2 h HBSS starvation) conditions. (H) Primary hepatocyte expressing Dyn2-GFP, showing an absence of colocalization with the LD surface (stained with Oil Red O). Bar, 20 µM. Inset shows magnification of boxed region (bar, 2 µM).
Dynasore, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 153 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/dynasore/product/Millipore
Average 99 stars, based on 153 article reviews
Price from $9.99 to $1999.99
dynasore - by Bioz Stars, 2020-09
99/100 stars

Images

1) Product Images from "Lipid droplet breakdown requires Dynamin 2 for vesiculation of autolysosomal tubules in hepatocytes"

Article Title: Lipid droplet breakdown requires Dynamin 2 for vesiculation of autolysosomal tubules in hepatocytes

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201306140

Pharmacological and genetic inhibition of Dyn2 function also reduces starvation-induced LD breakdown. HuH-7 (A) and Hep3B (B) hepatocytes were loaded with 150 µM oleate overnight and starved for 48 h in medium containing 0.1% FBS in the presence of Dyn2 inhibitors or DMSO as indicated. Representative images of inhibitor-treated and control cells (stained with Oil Red O) are shown in A and B together with the quantitation of the average LD area per cell from three independent experiments. Pharmacological inhibitors used were: Dynasore (inhibits Dyn2 GTPase activity), MiTMAB (targets PH domain and interferes with membrane binding), Dynole 34-2 (allosteric GTPase inhibitor), and Dynole 31-2 (negative control for Dynole 34-2). Bars, 20 µM. (C) Representative images from control and Dyn2 knockout MEFs after an overnight lipid loading with 400 µM oleate for 17 h. Knockout of Dyn2 was induced by treatment with 2 µM 4-hydroxy-tamoxifen for 7 d and was confirmed by immunostaining of endogenous Dyn2 (top row) and by immunoblot (D). Bars, 20 µM. (E and F) Average LD number (E) and area (F) per cell from five independent experiments. All data are represented as mean ± SE. *, P ≤ 0.05; **, P ≤ 0.01. (G) Whole-cell lysates and LD fractions isolated from HuH-7 hepatocytes under resting or starved (2 h HBSS starvation) conditions. (H) Primary hepatocyte expressing Dyn2-GFP, showing an absence of colocalization with the LD surface (stained with Oil Red O). Bar, 20 µM. Inset shows magnification of boxed region (bar, 2 µM).
Figure Legend Snippet: Pharmacological and genetic inhibition of Dyn2 function also reduces starvation-induced LD breakdown. HuH-7 (A) and Hep3B (B) hepatocytes were loaded with 150 µM oleate overnight and starved for 48 h in medium containing 0.1% FBS in the presence of Dyn2 inhibitors or DMSO as indicated. Representative images of inhibitor-treated and control cells (stained with Oil Red O) are shown in A and B together with the quantitation of the average LD area per cell from three independent experiments. Pharmacological inhibitors used were: Dynasore (inhibits Dyn2 GTPase activity), MiTMAB (targets PH domain and interferes with membrane binding), Dynole 34-2 (allosteric GTPase inhibitor), and Dynole 31-2 (negative control for Dynole 34-2). Bars, 20 µM. (C) Representative images from control and Dyn2 knockout MEFs after an overnight lipid loading with 400 µM oleate for 17 h. Knockout of Dyn2 was induced by treatment with 2 µM 4-hydroxy-tamoxifen for 7 d and was confirmed by immunostaining of endogenous Dyn2 (top row) and by immunoblot (D). Bars, 20 µM. (E and F) Average LD number (E) and area (F) per cell from five independent experiments. All data are represented as mean ± SE. *, P ≤ 0.05; **, P ≤ 0.01. (G) Whole-cell lysates and LD fractions isolated from HuH-7 hepatocytes under resting or starved (2 h HBSS starvation) conditions. (H) Primary hepatocyte expressing Dyn2-GFP, showing an absence of colocalization with the LD surface (stained with Oil Red O). Bar, 20 µM. Inset shows magnification of boxed region (bar, 2 µM).

Techniques Used: Inhibition, Staining, Quantitation Assay, Activity Assay, Binding Assay, Negative Control, Knock-Out, Immunostaining, Isolation, Expressing

Dyn2 associates with autolysosomal tubules. (A and B) Subcellular density gradient fractionation of Hep3B hepatocytes starved for 2 h in HBSS and treated with 40 µM Dynasore, to induce tubule formation, as in Fig. 5 . Cells were lysed (WCL), and the post-nuclear supernatant (PNS) was pelleted by centrifugation to produce a crude lysosomal fraction (CLF) and high-speed supernatant (HSS). The CLF was subsequently loaded onto an 8–27% discontinuous iodixanol (OptiPrep) gradient for separation by ultracentrifugation. Nine fractions were collected from the top of the gradient and blotted for Dyn2 and the lysosomal resident protein, LAMP1. Lysosomal acid phosphatase activity roughly correlates with LAMP1 protein levels in each fraction. Levels of Dyn2 are highest in fraction 2, corresponding with both the peak levels of LAMP1 and lysosomal activity. (A) The data shown are from a single representative experiment out of three repeats. (B) Blotting for subcellular components shows that Dyn2 is specific for these same fractions, unlike other organelle markers such as EEA1 (early endosomes) and COXIV (mitochondria). (C and D) Dyn2 localizes to the surface of LAMP1-positive compartments. Fluorescence imaging of Hep3B hepatocytes transfected with Dyn2-GFP and LAMP1-mCherry under resting (C and C′) or starvation (D) conditions. (C′′) Dyn2-GFP localizing to LAMP1-labeled lysosome structures under starvation conditions. Arrows indicate regions of protein colocalization. Dyn2 (arrows) is present at the site of scission of LAMP1-positive tubules (arrowhead) from large autolysosomal structures. Bars: (C) 5 µM; (C′, C′′, and D) 3 µM.
Figure Legend Snippet: Dyn2 associates with autolysosomal tubules. (A and B) Subcellular density gradient fractionation of Hep3B hepatocytes starved for 2 h in HBSS and treated with 40 µM Dynasore, to induce tubule formation, as in Fig. 5 . Cells were lysed (WCL), and the post-nuclear supernatant (PNS) was pelleted by centrifugation to produce a crude lysosomal fraction (CLF) and high-speed supernatant (HSS). The CLF was subsequently loaded onto an 8–27% discontinuous iodixanol (OptiPrep) gradient for separation by ultracentrifugation. Nine fractions were collected from the top of the gradient and blotted for Dyn2 and the lysosomal resident protein, LAMP1. Lysosomal acid phosphatase activity roughly correlates with LAMP1 protein levels in each fraction. Levels of Dyn2 are highest in fraction 2, corresponding with both the peak levels of LAMP1 and lysosomal activity. (A) The data shown are from a single representative experiment out of three repeats. (B) Blotting for subcellular components shows that Dyn2 is specific for these same fractions, unlike other organelle markers such as EEA1 (early endosomes) and COXIV (mitochondria). (C and D) Dyn2 localizes to the surface of LAMP1-positive compartments. Fluorescence imaging of Hep3B hepatocytes transfected with Dyn2-GFP and LAMP1-mCherry under resting (C and C′) or starvation (D) conditions. (C′′) Dyn2-GFP localizing to LAMP1-labeled lysosome structures under starvation conditions. Arrows indicate regions of protein colocalization. Dyn2 (arrows) is present at the site of scission of LAMP1-positive tubules (arrowhead) from large autolysosomal structures. Bars: (C) 5 µM; (C′, C′′, and D) 3 µM.

Techniques Used: Fractionation, Centrifugation, Activity Assay, Fluorescence, Imaging, Transfection, Labeling

Dyn2 inhibition leads to enlarged autolysosomal structures and prevents the autophagy of lipid droplets. Hep3B cells treated with either a nontargeting control siRNA (A and B, siNT) or an siRNA targeting human Dyn2 (C and D, siDyn2) were fixed and co-stained with antibodies specific for LAMP1 (red) and LC3 (green). After Dyn2 knockdown, a juxtanuclear aggregation and enlargement of the LAMP1-positive compartment is observed (C and D, arrows). Increased labeling of LC3 is also detectable after knockdown of Dyn2. (E) Western blotting of Hep3B lysates after a 3-d treatment with either the control or Dyn2-targeted siRNA and further treatment with or without 50 µM leupeptin. Densitometry-based analysis of six independent experiments is shown at the bottom of the figure. (F) Western blotting of Hep3B lysates after treatment for 2 h with DMSO or 80 µM Dynasore, in the presence or absence of 50 µM leupeptin. Quantitation of LC3-II levels relative to control are shown below the blots. The data are represented as mean ± SE; *, P ≤ 0.05.
Figure Legend Snippet: Dyn2 inhibition leads to enlarged autolysosomal structures and prevents the autophagy of lipid droplets. Hep3B cells treated with either a nontargeting control siRNA (A and B, siNT) or an siRNA targeting human Dyn2 (C and D, siDyn2) were fixed and co-stained with antibodies specific for LAMP1 (red) and LC3 (green). After Dyn2 knockdown, a juxtanuclear aggregation and enlargement of the LAMP1-positive compartment is observed (C and D, arrows). Increased labeling of LC3 is also detectable after knockdown of Dyn2. (E) Western blotting of Hep3B lysates after a 3-d treatment with either the control or Dyn2-targeted siRNA and further treatment with or without 50 µM leupeptin. Densitometry-based analysis of six independent experiments is shown at the bottom of the figure. (F) Western blotting of Hep3B lysates after treatment for 2 h with DMSO or 80 µM Dynasore, in the presence or absence of 50 µM leupeptin. Quantitation of LC3-II levels relative to control are shown below the blots. The data are represented as mean ± SE; *, P ≤ 0.05.

Techniques Used: Inhibition, Staining, Labeling, Western Blot, Quantitation Assay

Acute inhibition of Dyn2 reversibly disrupts autophagic lysosomal reformation (ALR) and lysosomal tubule scission. (A–D) Still frames from time-lapse movies of Hep3B cells expressing LAMP1-mCherry. Cells were starved for 2 h in HBSS and subsequently treated for 30 min with either DMSO (A and B) or 40 µM Dynasore (C and D), which induced extensive tubulation of LAMP1-positive compartments. Bars (A–D): 20 µM; (A′–B′) 2 µM; (C′–D′) 10 µM. (E–G) To demonstrate the reversibility of this tubulation, Dynasore-treated cells were washed extensively with drug-free media containing 10% FBS and monitored by time-lapse microscopy for 45 min. Frequently, after drug washout, LAMP1-positive tubules exhibited noticeable varicosities (E and F, arrows; bars, 10 µM) along their length. These sites are suggestive of areas of scission and resumed budding of nascent protolysosomes from the reformation tubules (G; bars, 10 µM). (H) Tubules from cells undergoing drug washout were quantified by tracing their lengths at the beginning and end of these movies. Still frames from a representative movie show tubule content at t = 10 and 45 min after drug washout. Bars, 20 µM. (I) Analysis of five independent movies showed an average decrease in total tubulation of ∼50% after drug washout. Data represent the average relative change in total tubule length between the first and last frames of the time-lapse movies. Error bars represent SE; *, P
Figure Legend Snippet: Acute inhibition of Dyn2 reversibly disrupts autophagic lysosomal reformation (ALR) and lysosomal tubule scission. (A–D) Still frames from time-lapse movies of Hep3B cells expressing LAMP1-mCherry. Cells were starved for 2 h in HBSS and subsequently treated for 30 min with either DMSO (A and B) or 40 µM Dynasore (C and D), which induced extensive tubulation of LAMP1-positive compartments. Bars (A–D): 20 µM; (A′–B′) 2 µM; (C′–D′) 10 µM. (E–G) To demonstrate the reversibility of this tubulation, Dynasore-treated cells were washed extensively with drug-free media containing 10% FBS and monitored by time-lapse microscopy for 45 min. Frequently, after drug washout, LAMP1-positive tubules exhibited noticeable varicosities (E and F, arrows; bars, 10 µM) along their length. These sites are suggestive of areas of scission and resumed budding of nascent protolysosomes from the reformation tubules (G; bars, 10 µM). (H) Tubules from cells undergoing drug washout were quantified by tracing their lengths at the beginning and end of these movies. Still frames from a representative movie show tubule content at t = 10 and 45 min after drug washout. Bars, 20 µM. (I) Analysis of five independent movies showed an average decrease in total tubulation of ∼50% after drug washout. Data represent the average relative change in total tubule length between the first and last frames of the time-lapse movies. Error bars represent SE; *, P

Techniques Used: Inhibition, Expressing, Time-lapse Microscopy

2) Product Images from "Ebolavirus Is Internalized into Host Cells via Macropinocytosis in a Viral Glycoprotein-Dependent Manner"

Article Title: Ebolavirus Is Internalized into Host Cells via Macropinocytosis in a Viral Glycoprotein-Dependent Manner

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1001121

Internalization of DiI-labeled EBOV particles is independent of the caveolae-mediated endocytic pathway. (A) DiI-labeled EBOV particles do not co-localize with eGFP-labeled caveolae. DiI-EbolaΔVP30 virions (left panel) or DiI-Ebola VLPs (right panel) were adsorbed to Cav1-eGFP-expressing Vero cells for 30 min on ice. The cells were then incubated for 15 min at 37°C and the co-localization of DiI-labeled viral particles with eGFP-labeled caveolae was analyzed by using confocal laser scanning microscope. Insets show enlargements of the boxed areas. Scale bars, 10 µm. (B) Effect of Cav1 down-regulation on the internalization of DiI-labeled Ebola virions. Vero cells were transfected with control siRNA (left panels) or siRNA to down-regulate Cav1 expression (right panels). The efficiency of Cav1 down-regulation was analyzed by use of immunofluorescent staining 48 h post-transfection (lower panels) and western blot analysis (C). Labeled Ebola VLPs were adsorbed to the siRNA-transfected cells for 30 min on ice 48 h post-transfection. After incubation for 2 h at 37°C, surface-bound virions were removed by the addition of trypsin for 5 min at 37°C and the internalization of Ebola VLPs was analyzed by using confocal laser scanning microscope (upper panels). Outlines of individual cells were drawn. Scale bars, 10 µm. (D) Quantitative analysis of the internalization of DiI-labeled Ebola virions in siRNA-transfected Vero cells. The internalized DiI-virions were analyzed in 10 individual siRNA-transfected cells. Each experiment was performed in triplicate and the results are presented as the mean ± SD. (E) Internalization of DiI-labeled Ebola virions in cells lacking Cav1. DiI-labeled EbolaΔVP30 virions were adsorbed to Cav1-deficient Huh7 cells for 30 min on ice. The internalization of DiI-EbolaΔVP30 virions was analyzed 2 h after the temperature shift to 37°C. Outlines of individual cells were drawn. Scale bar, 10 µm. (F) Effect of dynasore on the internalization of DiI-labeled Ebola virions. Vero cells were treated with DMSO (left panel) or dynasore (right panel) for 30 min at 37°C. Labeled Ebola VLPs were adsorbed to the cells for 30 min on ice and incubated for 2 h at 37°C in the presence of DMSO or dynasore. Surface-bound virions were removed by trypsin and the internalization of DiI-virions was analyzed by using confocal laser scanning microscope. Dynasore treatment interfered with the internalization of Alexa Fluor 633-Tf (green in right panel), attesting to its functionality. Scale bars, 10 µm. (G) Quantitative analysis of the internalization of DiI-labeled Ebola virions in dynasore-treated Vero cells. The internalized DiI-virions were analyzed in 10 individual DMSO- or dynasore-treated cells. Each experiment was performed in triplicate and the results are presented as the mean ± SD.
Figure Legend Snippet: Internalization of DiI-labeled EBOV particles is independent of the caveolae-mediated endocytic pathway. (A) DiI-labeled EBOV particles do not co-localize with eGFP-labeled caveolae. DiI-EbolaΔVP30 virions (left panel) or DiI-Ebola VLPs (right panel) were adsorbed to Cav1-eGFP-expressing Vero cells for 30 min on ice. The cells were then incubated for 15 min at 37°C and the co-localization of DiI-labeled viral particles with eGFP-labeled caveolae was analyzed by using confocal laser scanning microscope. Insets show enlargements of the boxed areas. Scale bars, 10 µm. (B) Effect of Cav1 down-regulation on the internalization of DiI-labeled Ebola virions. Vero cells were transfected with control siRNA (left panels) or siRNA to down-regulate Cav1 expression (right panels). The efficiency of Cav1 down-regulation was analyzed by use of immunofluorescent staining 48 h post-transfection (lower panels) and western blot analysis (C). Labeled Ebola VLPs were adsorbed to the siRNA-transfected cells for 30 min on ice 48 h post-transfection. After incubation for 2 h at 37°C, surface-bound virions were removed by the addition of trypsin for 5 min at 37°C and the internalization of Ebola VLPs was analyzed by using confocal laser scanning microscope (upper panels). Outlines of individual cells were drawn. Scale bars, 10 µm. (D) Quantitative analysis of the internalization of DiI-labeled Ebola virions in siRNA-transfected Vero cells. The internalized DiI-virions were analyzed in 10 individual siRNA-transfected cells. Each experiment was performed in triplicate and the results are presented as the mean ± SD. (E) Internalization of DiI-labeled Ebola virions in cells lacking Cav1. DiI-labeled EbolaΔVP30 virions were adsorbed to Cav1-deficient Huh7 cells for 30 min on ice. The internalization of DiI-EbolaΔVP30 virions was analyzed 2 h after the temperature shift to 37°C. Outlines of individual cells were drawn. Scale bar, 10 µm. (F) Effect of dynasore on the internalization of DiI-labeled Ebola virions. Vero cells were treated with DMSO (left panel) or dynasore (right panel) for 30 min at 37°C. Labeled Ebola VLPs were adsorbed to the cells for 30 min on ice and incubated for 2 h at 37°C in the presence of DMSO or dynasore. Surface-bound virions were removed by trypsin and the internalization of DiI-virions was analyzed by using confocal laser scanning microscope. Dynasore treatment interfered with the internalization of Alexa Fluor 633-Tf (green in right panel), attesting to its functionality. Scale bars, 10 µm. (G) Quantitative analysis of the internalization of DiI-labeled Ebola virions in dynasore-treated Vero cells. The internalized DiI-virions were analyzed in 10 individual DMSO- or dynasore-treated cells. Each experiment was performed in triplicate and the results are presented as the mean ± SD.

Techniques Used: Labeling, Expressing, Incubation, Laser-Scanning Microscopy, Transfection, Staining, Western Blot

3) Product Images from "Alternative infectious entry pathways for dengue virus serotypes into mammalian cells"

Article Title: Alternative infectious entry pathways for dengue virus serotypes into mammalian cells

Journal: Cellular Microbiology

doi: 10.1111/j.1462-5822.2009.01345.x

DENV‐2 entry into Vero cells is independent of caveolae/lipid‐rafts but is dependent on dynamin. A. DENV‐1 or DENV‐2 suspensions were incubated at 37°C with various concentrations of methyl‐β‐cyclodextrin. After 1 h remaining infectivity was determined. B. Vero cells were pretreated with nystatin or methyl‐β‐cyclodextrin. Then monolayers were washed with PBS and infected with DENV‐1 or DENV‐2 in culture medium without serum. After 1 h internalization, cultures were treated with proteinase K and the cell pellets were plated onto Vero cells to determine internalized virus by an infectious centre assay. Results are expressed as percentage of internalized virus with respect to a control without drug treatment. C. Cells were untreated (control) or treated with 100 μM nystatin or 5 mM methyl‐β‐cyclodextrin. Then cultures were incubated with FITC‐labelled cholera toxin. D. Cells transiently transfected with the constructs GFP‐cav‐1 wt, GFP cav‐1 DN or GFP‐cav‐1 Y14F were infected with DENV‐2. After 24 h infection cultures were fixed and immunofluorescence staining was performed. E. For quantification of samples shown in (D), 250 transfected cells with similar levels of GFP expression were screened and cells positive for viral antigen were scored. F. Vero cells were treated with dynasore, infected with DENV‐1 or DENV‐2 in the presence of the drug and then processed as in (B). G. Cells transiently transfected with the constructs GFP‐dyn II wt or GFP‐dyn II K44A were infected with DENV‐2. After 24 h infection cultures were fixed and immunofluorescence staining was performed. H. For quantification of samples shown in (G), 250 transfected cells with similar levels of GFP expression were screened and cells positive for viral antigen were scored. In (A), (B), (E), (F) and (H) values represent the mean ± SD of two independent experiments.
Figure Legend Snippet: DENV‐2 entry into Vero cells is independent of caveolae/lipid‐rafts but is dependent on dynamin. A. DENV‐1 or DENV‐2 suspensions were incubated at 37°C with various concentrations of methyl‐β‐cyclodextrin. After 1 h remaining infectivity was determined. B. Vero cells were pretreated with nystatin or methyl‐β‐cyclodextrin. Then monolayers were washed with PBS and infected with DENV‐1 or DENV‐2 in culture medium without serum. After 1 h internalization, cultures were treated with proteinase K and the cell pellets were plated onto Vero cells to determine internalized virus by an infectious centre assay. Results are expressed as percentage of internalized virus with respect to a control without drug treatment. C. Cells were untreated (control) or treated with 100 μM nystatin or 5 mM methyl‐β‐cyclodextrin. Then cultures were incubated with FITC‐labelled cholera toxin. D. Cells transiently transfected with the constructs GFP‐cav‐1 wt, GFP cav‐1 DN or GFP‐cav‐1 Y14F were infected with DENV‐2. After 24 h infection cultures were fixed and immunofluorescence staining was performed. E. For quantification of samples shown in (D), 250 transfected cells with similar levels of GFP expression were screened and cells positive for viral antigen were scored. F. Vero cells were treated with dynasore, infected with DENV‐1 or DENV‐2 in the presence of the drug and then processed as in (B). G. Cells transiently transfected with the constructs GFP‐dyn II wt or GFP‐dyn II K44A were infected with DENV‐2. After 24 h infection cultures were fixed and immunofluorescence staining was performed. H. For quantification of samples shown in (G), 250 transfected cells with similar levels of GFP expression were screened and cells positive for viral antigen were scored. In (A), (B), (E), (F) and (H) values represent the mean ± SD of two independent experiments.

Techniques Used: Incubation, Infection, Transfection, Construct, Immunofluorescence, Staining, Expressing

4) Product Images from "Macropinocytic entry of isolated mitochondria in epidermal growth factor-activated human osteosarcoma cells"

Article Title: Macropinocytic entry of isolated mitochondria in epidermal growth factor-activated human osteosarcoma cells

Journal: Scientific Reports

doi: 10.1038/s41598-017-13227-0

Internalisation of mitochondria occurs by endocytosis in HOS cells, with a macropinocytic activator EGF upregulating the process. ( A – F ) Internalisation of EGFP-labelled mitochondria in HOS cells, as quantified by FACS, in the presence and absence of the endocytic inhibitors: chlorpromazine (CPZ) (100 μm) ( A ), MβCD (5 mM) ( B ), dynasore (120 μm) ( C ), EIPA (50 μm) ( D ), wortmannin (300 nM) ( E ) and latrunculin A (0.5 µM) ( F ). ( G , H ) EGF upregulates mitochondrial internalisation. Internalisation of EGFP-labelled mitochondria by HOS cells, as quantified by FACS, in the presence and absence of 50 nM and 100 nM EGF ( G ), 50 nM FGF and 100 nM FGF ( H ). The Y axis shows the percentage of green fluorescent cells normalised to the numbers of green cells in a control sample (cells incubated with only EGFP-labelled mitochondria for 90 min). The number of green fluorescent cells in this control sample was taken to represent 100%. The green fluorescence in all samples for inhibitor assays of mitochondrial internalisation were normalised to levels in their respective control samples. Data shown as mean values +/− s.e.m. n = 3 ( A – D and F – H ) n = 6 ( E ). *p
Figure Legend Snippet: Internalisation of mitochondria occurs by endocytosis in HOS cells, with a macropinocytic activator EGF upregulating the process. ( A – F ) Internalisation of EGFP-labelled mitochondria in HOS cells, as quantified by FACS, in the presence and absence of the endocytic inhibitors: chlorpromazine (CPZ) (100 μm) ( A ), MβCD (5 mM) ( B ), dynasore (120 μm) ( C ), EIPA (50 μm) ( D ), wortmannin (300 nM) ( E ) and latrunculin A (0.5 µM) ( F ). ( G , H ) EGF upregulates mitochondrial internalisation. Internalisation of EGFP-labelled mitochondria by HOS cells, as quantified by FACS, in the presence and absence of 50 nM and 100 nM EGF ( G ), 50 nM FGF and 100 nM FGF ( H ). The Y axis shows the percentage of green fluorescent cells normalised to the numbers of green cells in a control sample (cells incubated with only EGFP-labelled mitochondria for 90 min). The number of green fluorescent cells in this control sample was taken to represent 100%. The green fluorescence in all samples for inhibitor assays of mitochondrial internalisation were normalised to levels in their respective control samples. Data shown as mean values +/− s.e.m. n = 3 ( A – D and F – H ) n = 6 ( E ). *p

Techniques Used: FACS, Incubation, Fluorescence

5) Product Images from "Blockade of dengue virus entry into myeloid cells by endocytic inhibitors in the presence or absence of antibodies"

Article Title: Blockade of dengue virus entry into myeloid cells by endocytic inhibitors in the presence or absence of antibodies

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0006685

Effect of the endocytosis inhibitors on the Ab-mediated infection of U937and K562 cells. U937 (black bars) or K562 (grey bars) cells were treated with ammonium chloride (A), chlorpromazine (B), dansylcadaverine (C), dynasore (D) or methyl-β-cyclodextrin (E), and then infected with the complexes DENV-2-2H2 or DENV-2-3H5, respectively. At 72 h p.i., the virus yields were determined by plaque formation in Vero cells and the results are expressed as % of virus multiplication with respect to a control of complex infected cells without drug treatment. Each bar is the mean of three independent experiments ± SEM. Asterisks indicate statistical significance between treated and control infected cells (** P
Figure Legend Snippet: Effect of the endocytosis inhibitors on the Ab-mediated infection of U937and K562 cells. U937 (black bars) or K562 (grey bars) cells were treated with ammonium chloride (A), chlorpromazine (B), dansylcadaverine (C), dynasore (D) or methyl-β-cyclodextrin (E), and then infected with the complexes DENV-2-2H2 or DENV-2-3H5, respectively. At 72 h p.i., the virus yields were determined by plaque formation in Vero cells and the results are expressed as % of virus multiplication with respect to a control of complex infected cells without drug treatment. Each bar is the mean of three independent experiments ± SEM. Asterisks indicate statistical significance between treated and control infected cells (** P

Techniques Used: Infection

Time-dependence of the antiviral activity of the endocytic inhibitors. Cells were treated with 50 mM ammonium chloride (A), 20 μM chlorpromazine (B) or 160 μM dynasore (C) before infection with DENV-2 (pretreatment) or after 2 h of infection with DENV-2 (post internalization) at a m.o.i. of 5 PFU/cell. At 48 h p.i. the extracellular virus yields were determined by plaque formation in Vero cells and the results are expressed as % of virus yield in treated infected cells with respect to a control of infected cells without drug treatment. Each bar is the mean of three independent experiments ± SEM.
Figure Legend Snippet: Time-dependence of the antiviral activity of the endocytic inhibitors. Cells were treated with 50 mM ammonium chloride (A), 20 μM chlorpromazine (B) or 160 μM dynasore (C) before infection with DENV-2 (pretreatment) or after 2 h of infection with DENV-2 (post internalization) at a m.o.i. of 5 PFU/cell. At 48 h p.i. the extracellular virus yields were determined by plaque formation in Vero cells and the results are expressed as % of virus yield in treated infected cells with respect to a control of infected cells without drug treatment. Each bar is the mean of three independent experiments ± SEM.

Techniques Used: Activity Assay, Infection

Control of the endocytosis inhibitors function. U937 and K562 cells were treated with 50 mM ammonium chloride (A), 2.5 mM methyl-β-cyclodextrin (B), 20 μM chlorpromazine, 150 μM dansylcadaverine, 160 μM dynasore (C) or untreated (control) and then incubated with acridine orange (A), FITC-labelled cholera toxin (B) or TRITC-labelled transferrin (C). Controls without drugs were performed and the samples were visualized by fluorescence microscope.
Figure Legend Snippet: Control of the endocytosis inhibitors function. U937 and K562 cells were treated with 50 mM ammonium chloride (A), 2.5 mM methyl-β-cyclodextrin (B), 20 μM chlorpromazine, 150 μM dansylcadaverine, 160 μM dynasore (C) or untreated (control) and then incubated with acridine orange (A), FITC-labelled cholera toxin (B) or TRITC-labelled transferrin (C). Controls without drugs were performed and the samples were visualized by fluorescence microscope.

Techniques Used: Incubation, Fluorescence, Microscopy

Effect of the endocytosis inhibitors on DENV-2 infection determined by quantitative RT-PCR. U937 (black bars) or K562 (grey bars) cells were treated with ammonium chloride (A), chlorpromazine (B), dynasore (C) or methyl-β-cyclodextrin (D), and then infected with DENV-2. At 12 h p.i., the total RNA was extracted from the cells with TRIzol and the amount of viral RNA molecules was measured by quantitative RT-PCR. Each bar is the mean of three independent experiments ± SEM. Asterisks indicate statistical significance between treated and control infected cells (** P
Figure Legend Snippet: Effect of the endocytosis inhibitors on DENV-2 infection determined by quantitative RT-PCR. U937 (black bars) or K562 (grey bars) cells were treated with ammonium chloride (A), chlorpromazine (B), dynasore (C) or methyl-β-cyclodextrin (D), and then infected with DENV-2. At 12 h p.i., the total RNA was extracted from the cells with TRIzol and the amount of viral RNA molecules was measured by quantitative RT-PCR. Each bar is the mean of three independent experiments ± SEM. Asterisks indicate statistical significance between treated and control infected cells (** P

Techniques Used: Infection, Quantitative RT-PCR

Effect of the endocytosis inhibitors on DENV-2 infective virus production in U937 and K562 cells. U937 (black bars) or K562 (grey bars) cells were treated with ammonium chloride (A), chlorpromazine (B), dynasore (C) or methyl-β-cyclodextrin (D), and then infected with DENV-2. At 48 h p.i., the virus yields were determined by plaque formation in Vero cells and the results are expressed as % of virus multiplication with respect to a control of infected cells without drug treatment. Each bar is the mean of three independent experiments ± SEM. Asterisks indicate statistical significance between treated and control infected cells (** P
Figure Legend Snippet: Effect of the endocytosis inhibitors on DENV-2 infective virus production in U937 and K562 cells. U937 (black bars) or K562 (grey bars) cells were treated with ammonium chloride (A), chlorpromazine (B), dynasore (C) or methyl-β-cyclodextrin (D), and then infected with DENV-2. At 48 h p.i., the virus yields were determined by plaque formation in Vero cells and the results are expressed as % of virus multiplication with respect to a control of infected cells without drug treatment. Each bar is the mean of three independent experiments ± SEM. Asterisks indicate statistical significance between treated and control infected cells (** P

Techniques Used: Infection

6) 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

7) Product Images from "Kaposi's Sarcoma-Associated Herpesvirus Utilizes an Actin Polymerization-Dependent Macropinocytic Pathway To Enter Human Dermal Microvascular Endothelial and Human Umbilical Vein Endothelial Cells ▿"

Article Title: Kaposi's Sarcoma-Associated Herpesvirus Utilizes an Actin Polymerization-Dependent Macropinocytic Pathway To Enter Human Dermal Microvascular Endothelial and Human Umbilical Vein Endothelial Cells ▿

Journal: Journal of Virology

doi: 10.1128/JVI.02498-08

KSHV entry in HMVEC-d and HUVEC cells is dynamin independent. (A) HMVEC-d cells showing transfection with WT dynamin. Cells were transfected with 1 μg of GFP-WT dynamin plasmid or 1 μg of GFP-K44A dynamin plasmid and observed under a microscope after 24 h. a and c, GFP; b and d, phase-contrast microscopy. Magnification, ×10. (B) HMVEC-d cells were transfected with 1 μg of GFP-WT or GFP-K44A dynamin plasmid. After 24 h, cells were infected with KSHV at an MOI of 10 for different times and KSHV internalization was measured by real-time DNA PCR. Histogram shows internalized copy numbers of KSHV in HMVEC-d cells transfected with WT and K44A dynamin plasmids. (C and D) Dynasore did not affect internalization in HUVEC (C) and HMVEC-d (D) cells. HUVEC cells and HMVEC-d cells grown in six-well plates were treated with 80 μM dynasore for 1 h at 37°C and infected with KSHV at an MOI of 10 for different times, and KSHV internalization measured by real-time DNA PCR for ORF73 gene. (E) Dynasore treatment affects KSHV internalization in HFF cells. Cells grown in six-well plates were treated with 100 μM dynasore and infected with KSHV at an MOI of 10 for different times, and KSHV internalization measured by real-time DNA PCR. Histogram shows the internalized copy numbers of KSHV DNA with and without dynasore treatment. Each reaction was done in duplicate, and each bar represents the mean ± standard deviation of the results of three experiments.
Figure Legend Snippet: KSHV entry in HMVEC-d and HUVEC cells is dynamin independent. (A) HMVEC-d cells showing transfection with WT dynamin. Cells were transfected with 1 μg of GFP-WT dynamin plasmid or 1 μg of GFP-K44A dynamin plasmid and observed under a microscope after 24 h. a and c, GFP; b and d, phase-contrast microscopy. Magnification, ×10. (B) HMVEC-d cells were transfected with 1 μg of GFP-WT or GFP-K44A dynamin plasmid. After 24 h, cells were infected with KSHV at an MOI of 10 for different times and KSHV internalization was measured by real-time DNA PCR. Histogram shows internalized copy numbers of KSHV in HMVEC-d cells transfected with WT and K44A dynamin plasmids. (C and D) Dynasore did not affect internalization in HUVEC (C) and HMVEC-d (D) cells. HUVEC cells and HMVEC-d cells grown in six-well plates were treated with 80 μM dynasore for 1 h at 37°C and infected with KSHV at an MOI of 10 for different times, and KSHV internalization measured by real-time DNA PCR for ORF73 gene. (E) Dynasore treatment affects KSHV internalization in HFF cells. Cells grown in six-well plates were treated with 100 μM dynasore and infected with KSHV at an MOI of 10 for different times, and KSHV internalization measured by real-time DNA PCR. Histogram shows the internalized copy numbers of KSHV DNA with and without dynasore treatment. Each reaction was done in duplicate, and each bar represents the mean ± standard deviation of the results of three experiments.

Techniques Used: Transfection, Plasmid Preparation, Microscopy, Infection, Polymerase Chain Reaction, Standard Deviation

8) Product Images from "Clathrin Pit-mediated Endocytosis of Neutrophil Elastase and Cathepsin G by Cancer Cells *"

Article Title: Clathrin Pit-mediated Endocytosis of Neutrophil Elastase and Cathepsin G by Cancer Cells *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M112.385617

CG enters tumor endosomes. A549 cells were incubated with Alexa Fluor 488-labeled CG for 30 min either alone ( A ), or with the addition of PMSF ( B ), heat (100 ºC) ( C ), urea ( D ), guanidine ( E ), dynasore ( F ), scrambled siRNA ( H ), heavy chain clathrin
Figure Legend Snippet: CG enters tumor endosomes. A549 cells were incubated with Alexa Fluor 488-labeled CG for 30 min either alone ( A ), or with the addition of PMSF ( B ), heat (100 ºC) ( C ), urea ( D ), guanidine ( E ), dynasore ( F ), scrambled siRNA ( H ), heavy chain clathrin

Techniques Used: Incubation, Labeling

NE endocytosis is required for cellular proliferation. A and B , 3 H uptake for H23 cells treated with NE (0–40 n m ) for 60 min in the absence ( A ) or presence ( B ) of dynasore. Results are shown as percentage of control. The experiment was done in
Figure Legend Snippet: NE endocytosis is required for cellular proliferation. A and B , 3 H uptake for H23 cells treated with NE (0–40 n m ) for 60 min in the absence ( A ) or presence ( B ) of dynasore. Results are shown as percentage of control. The experiment was done in

Techniques Used:

9) Product Images from "Dynamin Is Required for GnRH Signaling to L-Type Calcium Channels and Activation of ERK"

Article Title: Dynamin Is Required for GnRH Signaling to L-Type Calcium Channels and Activation of ERK

Journal: Endocrinology

doi: 10.1210/en.2015-1575

Dynamin facilitates GnRH-mediated ERK activation upstream of PKC. αT3–1 cells were pretreated with vehicle (0) or dynasore (80μM) for 30 minutes and then treated in the presence or absence of either GnRHa (10nM) or PMA (10nM) for
Figure Legend Snippet: Dynamin facilitates GnRH-mediated ERK activation upstream of PKC. αT3–1 cells were pretreated with vehicle (0) or dynasore (80μM) for 30 minutes and then treated in the presence or absence of either GnRHa (10nM) or PMA (10nM) for

Techniques Used: Activation Assay

Inhibition of dynamin decreases p-ERK in a dose-dependent manner. A, αT3–1 cells were pretreated (30 min) with vehicle (0) or the dynamin inhibitor dynasore (1μM, 40μM, 80μM, and 160μM) followed by a 10-minute
Figure Legend Snippet: Inhibition of dynamin decreases p-ERK in a dose-dependent manner. A, αT3–1 cells were pretreated (30 min) with vehicle (0) or the dynamin inhibitor dynasore (1μM, 40μM, 80μM, and 160μM) followed by a 10-minute

Techniques Used: Inhibition

Inhibition of dynamin GTPase activity decreases p-ERK activity in multiple gonadotrope cell lines. A, αT3–1 cells were pretreated (30 min) with vehicle (0), dynasore (80μM), or dyngo (30μM) for 30 minutes followed by a
Figure Legend Snippet: Inhibition of dynamin GTPase activity decreases p-ERK activity in multiple gonadotrope cell lines. A, αT3–1 cells were pretreated (30 min) with vehicle (0), dynasore (80μM), or dyngo (30μM) for 30 minutes followed by a

Techniques Used: Inhibition, Activity Assay

Inhibition of dynamin decreases GnRH-dependent Ca 2+ influx via L-type Ca 2+ channels in αT3–1 cells. A, Representative TIRF images showing localized Ca 2+ influx in αT3–1 cells treated with GnRH (3nM) with or without dynasore
Figure Legend Snippet: Inhibition of dynamin decreases GnRH-dependent Ca 2+ influx via L-type Ca 2+ channels in αT3–1 cells. A, Representative TIRF images showing localized Ca 2+ influx in αT3–1 cells treated with GnRH (3nM) with or without dynasore

Techniques Used: Inhibition

Dynamin inhibition blunts GnRHa-induced actin remodeling in αT3–1. A, αT3–1 were grown on glass-bottom microwell dishes for 24 hours. Cells were incubated in the presence and/or absence of 80μM dynasore for 30 minutes
Figure Legend Snippet: Dynamin inhibition blunts GnRHa-induced actin remodeling in αT3–1. A, αT3–1 were grown on glass-bottom microwell dishes for 24 hours. Cells were incubated in the presence and/or absence of 80μM dynasore for 30 minutes

Techniques Used: Inhibition, Incubation

10) Product Images from "Cathelicidin promotes inflammation by enabling binding of self-RNA to cell surface scavenger receptors"

Article Title: Cathelicidin promotes inflammation by enabling binding of self-RNA to cell surface scavenger receptors

Journal: Scientific Reports

doi: 10.1038/s41598-018-22409-3

Immune response to LL37 requires clathrin-dependent endocytosis ( a ) IL-6 mRNA in NHEKs cultured with various endocytosis inhibitors (Monodansylcadaverine (MDC): 200 μM, Pitstop-2 TM 25 μM, dynasore: 80 μM) for 30 minutes, treated with LL37 (2.5 μM) and U1 RNA (2.5 μg/mL) for a further 6 hours. (n = 3). ( b ) IL-6 mRNA in PMA-treated THP1 after treatment with cathelicidin peptides (3 μM) for 10 minutes, then stimulated with U1 RNA (12.5 μg/mL) overnight. (n = 3). ( c ) Gene sets induced in NHEK by LL37 + U1 RNA (fold change > 2 versus vehicle control) and genes of identically treated cells first repressed by Pitstop-2 TM (25 μM) (fold change
Figure Legend Snippet: Immune response to LL37 requires clathrin-dependent endocytosis ( a ) IL-6 mRNA in NHEKs cultured with various endocytosis inhibitors (Monodansylcadaverine (MDC): 200 μM, Pitstop-2 TM 25 μM, dynasore: 80 μM) for 30 minutes, treated with LL37 (2.5 μM) and U1 RNA (2.5 μg/mL) for a further 6 hours. (n = 3). ( b ) IL-6 mRNA in PMA-treated THP1 after treatment with cathelicidin peptides (3 μM) for 10 minutes, then stimulated with U1 RNA (12.5 μg/mL) overnight. (n = 3). ( c ) Gene sets induced in NHEK by LL37 + U1 RNA (fold change > 2 versus vehicle control) and genes of identically treated cells first repressed by Pitstop-2 TM (25 μM) (fold change

Techniques Used: Cell Culture

11) Product Images from "Quantifying the dynamic interactions between a clathrin-coated pit and cargo molecules"

Article Title: Quantifying the dynamic interactions between a clathrin-coated pit and cargo molecules

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

doi: 10.1073/pnas.1315202110

Binding-time distributions of Kv2.1. ( A ). ( B ) Schematic of ΔC-Kv2.1 mutant channel. The black tick marks where the C terminus (grayed) is removed. ( C ) Binding-time distribution for ΔC-Kv2.1. Without the C terminus, Kv2.1 becomes captured into CCPs less frequently, and it only remains bound for short times. ( D ) Binding-time distribution of Kv2.1 after inhibition of the GTPase dynamin with 80 μM dynasore. No statistically significant changes in the binding-time distribution are observed between control cells and dynasore-treated cells.
Figure Legend Snippet: Binding-time distributions of Kv2.1. ( A ). ( B ) Schematic of ΔC-Kv2.1 mutant channel. The black tick marks where the C terminus (grayed) is removed. ( C ) Binding-time distribution for ΔC-Kv2.1. Without the C terminus, Kv2.1 becomes captured into CCPs less frequently, and it only remains bound for short times. ( D ) Binding-time distribution of Kv2.1 after inhibition of the GTPase dynamin with 80 μM dynasore. No statistically significant changes in the binding-time distribution are observed between control cells and dynasore-treated cells.

Techniques Used: Binding Assay, Mutagenesis, Inhibition

Kv channels are internalized via CME. ( A ) Fraction of channels endocytosed in 10 min. The graph shows endocytic fraction for Kv2.1 and Kv1.4 with and without dynamin inhibitor dynasore. ( B ) Fluorescence time series of Kv2.1 endocytic event. The yellow arrow indicates QD-Kv2.1 (bottom row) and GFP-CCP (middle row) location. Both the fluorescence of the QD and GFP disappear at the same time, marking the endocytic event. (Scale bar: 1 μm.)
Figure Legend Snippet: Kv channels are internalized via CME. ( A ) Fraction of channels endocytosed in 10 min. The graph shows endocytic fraction for Kv2.1 and Kv1.4 with and without dynamin inhibitor dynasore. ( B ) Fluorescence time series of Kv2.1 endocytic event. The yellow arrow indicates QD-Kv2.1 (bottom row) and GFP-CCP (middle row) location. Both the fluorescence of the QD and GFP disappear at the same time, marking the endocytic event. (Scale bar: 1 μm.)

Techniques Used: Fluorescence

12) Product Images from "Endocytic uptake of monomeric amyloid-β peptides is clathrin- and dynamin-independent and results in selective accumulation of Aβ(1–42) compared to Aβ(1–40)"

Article Title: Endocytic uptake of monomeric amyloid-β peptides is clathrin- and dynamin-independent and results in selective accumulation of Aβ(1–42) compared to Aβ(1–40)

Journal: Scientific Reports

doi: 10.1038/s41598-017-02227-9

Uptake of Aβ(1–40), Aβ(1–42) and Trf in SH-SY5Y cells under conditions that perturb dynamin dependent endocytosis. ( a ) Uptake of HF488-labelled Aβ(1–40), Aβ(1–42) and AF647-labelled Trf in cells treated with dynasore (80 µM). The peptide uptake is reported as mean cellular uptake relative to control (cells not treated with inhibitor) for 4 independent experiments, each performed in triplicate. ( b ) Correlation analysis of the data presented in ( a ) with R 2 of the linear fit of average inhibitions levels from each experiment and treatment, and Pearson’s correlation coefficient (r). ( c ) Statistical analysis of the data in ( a ) performed by one-way ANOVA with matched data followed by multiple comparisons with Bonferroni post-hoc test. The table shows the adjusted p-values for the individual comparisons made (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001). ( d ) Mean cellular intensity ± SD (N = 2, n = 3–5) of live cells after incubation with HF647-labelled Aβ(1–40) or Aβ(1–42). ( e ) Flow cytometry histogram of live cells 24 h post transfection with EGFP-labelled dyn2 K44A: the cells were gated for peptide uptake based on transfection efficiency and the extent of dyn K44A overexpression as measured by the intensity of the EGFP label. ( f ) Uptake of HF647-labelled Aβ(1–40), Aβ(1–42) or AF647-labelled Trf in dyn2 K44A transfected cells, gated as in ( e ) and related to peptide uptake in cells gated as non-transfected (N = 2, n = 3–5). ( g – i ) Confocal imaging of dyn2 K44A (green) transfected cells imaged 24 h post transfection and incubated with HF647-labelled ( g ) Aβ(1–40), ( h ) Aβ(1–42) or ( i ) AF647-labelled Trf (red). In all experiments the concentration of Aβ was 1 μM and the concentration of Trf was 5 µg/ml. Cells were incubated with Aβ for 1 h and Trf for 5 min (flow cytometry) or 10 min (confocal microscopy). Relative uptake was calculated based on mean cellular fluorescence intensity ± SD of the total number of gated live cells measured by flow cytometry. All flow cytometry data were corrected for baseline contributions by subtracting the cellular autofluorescence.
Figure Legend Snippet: Uptake of Aβ(1–40), Aβ(1–42) and Trf in SH-SY5Y cells under conditions that perturb dynamin dependent endocytosis. ( a ) Uptake of HF488-labelled Aβ(1–40), Aβ(1–42) and AF647-labelled Trf in cells treated with dynasore (80 µM). The peptide uptake is reported as mean cellular uptake relative to control (cells not treated with inhibitor) for 4 independent experiments, each performed in triplicate. ( b ) Correlation analysis of the data presented in ( a ) with R 2 of the linear fit of average inhibitions levels from each experiment and treatment, and Pearson’s correlation coefficient (r). ( c ) Statistical analysis of the data in ( a ) performed by one-way ANOVA with matched data followed by multiple comparisons with Bonferroni post-hoc test. The table shows the adjusted p-values for the individual comparisons made (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001). ( d ) Mean cellular intensity ± SD (N = 2, n = 3–5) of live cells after incubation with HF647-labelled Aβ(1–40) or Aβ(1–42). ( e ) Flow cytometry histogram of live cells 24 h post transfection with EGFP-labelled dyn2 K44A: the cells were gated for peptide uptake based on transfection efficiency and the extent of dyn K44A overexpression as measured by the intensity of the EGFP label. ( f ) Uptake of HF647-labelled Aβ(1–40), Aβ(1–42) or AF647-labelled Trf in dyn2 K44A transfected cells, gated as in ( e ) and related to peptide uptake in cells gated as non-transfected (N = 2, n = 3–5). ( g – i ) Confocal imaging of dyn2 K44A (green) transfected cells imaged 24 h post transfection and incubated with HF647-labelled ( g ) Aβ(1–40), ( h ) Aβ(1–42) or ( i ) AF647-labelled Trf (red). In all experiments the concentration of Aβ was 1 μM and the concentration of Trf was 5 µg/ml. Cells were incubated with Aβ for 1 h and Trf for 5 min (flow cytometry) or 10 min (confocal microscopy). Relative uptake was calculated based on mean cellular fluorescence intensity ± SD of the total number of gated live cells measured by flow cytometry. All flow cytometry data were corrected for baseline contributions by subtracting the cellular autofluorescence.

Techniques Used: Incubation, Flow Cytometry, Cytometry, Transfection, Over Expression, Imaging, Concentration Assay, Confocal Microscopy, Fluorescence

13) Product Images from "Endosomal trafficking of the receptor tyrosine kinase MuSK proceeds via clathrin-dependent pathways, Arf6 and actin"

Article Title: Endosomal trafficking of the receptor tyrosine kinase MuSK proceeds via clathrin-dependent pathways, Arf6 and actin

Journal: The Febs Journal

doi: 10.1111/febs.12309

Dok7-dependent MuSK activation does not alter the rate of MuSK internalization but increases the recruitment into caveolin-positive structures on the cell surface. (A) To quantify the influence of Dok7 on the rate of MuSK endocytosis, COS-7 cells were transiently transfected with SBP-MuSK alone or together with Dok7. Surface MuSK was labelled with DyLight 649-conjugated streptavidin at 4 °C followed by incubation at 37 °C for different time periods. The remaining surface staining was stripped off and cells were harvested for intracellular fluorescence detection by FACS. A quantification of internalized MuSK is shown. Error bars indicate the SEM ( n = 3). (B) To determine whether the presence of Dok7 leads to a switch in the endocytic pathway of MuSK, COS-7 cells were transiently transfected with SBP-MuSK together with Dok7. Surface MuSK was labelled with Cy3-conjugated streptavidin at 4 °C followed by incubation at 37 °C for 5 min. After fixation, cells were stained with an antibody against the endogenous clathrin or caveolin. Quantification of colocalization using a threshold-and object-based colocalization analysis (as described in the Materials and methods) is shown. Error bars indicate the SEM ( n ≥ 12, from at least two independent experiments). (C) To quantify MuSK endocytosis in the presence or absence of inhibitors, COS-7 cells were transiently co-transfected with SBP-MuSK together with Dok7 and, as indicated, with AP180C, Eps15DIII or Dyn2K44A. Cells, treated with dimethylsulfoxide or dynasore, were stained with streptavidin conjugated-DyLight 649 followed by incubation at 37 °C for 5 min. The remaining surface staining was stripped off and cells were harvested for intracellular fluorescence detection by FACS. Internalized MuSK was quantified. The control sample denotes GFP-positive cells presenting a streptavidin signal. The dimethylsulfoxide sample represents streptavidin-positive cells treated with the solvent dimethylsulfoxide only. Error bars indicate the SEM ( n = 3). (D) COS-7 cells were transiently transfected with SBP-MuSK and GFP or Lrp4-GFP, respectively. MuSK internalization was detected as described in (A). Error bars indicate the SEM (n = 3).
Figure Legend Snippet: Dok7-dependent MuSK activation does not alter the rate of MuSK internalization but increases the recruitment into caveolin-positive structures on the cell surface. (A) To quantify the influence of Dok7 on the rate of MuSK endocytosis, COS-7 cells were transiently transfected with SBP-MuSK alone or together with Dok7. Surface MuSK was labelled with DyLight 649-conjugated streptavidin at 4 °C followed by incubation at 37 °C for different time periods. The remaining surface staining was stripped off and cells were harvested for intracellular fluorescence detection by FACS. A quantification of internalized MuSK is shown. Error bars indicate the SEM ( n = 3). (B) To determine whether the presence of Dok7 leads to a switch in the endocytic pathway of MuSK, COS-7 cells were transiently transfected with SBP-MuSK together with Dok7. Surface MuSK was labelled with Cy3-conjugated streptavidin at 4 °C followed by incubation at 37 °C for 5 min. After fixation, cells were stained with an antibody against the endogenous clathrin or caveolin. Quantification of colocalization using a threshold-and object-based colocalization analysis (as described in the Materials and methods) is shown. Error bars indicate the SEM ( n ≥ 12, from at least two independent experiments). (C) To quantify MuSK endocytosis in the presence or absence of inhibitors, COS-7 cells were transiently co-transfected with SBP-MuSK together with Dok7 and, as indicated, with AP180C, Eps15DIII or Dyn2K44A. Cells, treated with dimethylsulfoxide or dynasore, were stained with streptavidin conjugated-DyLight 649 followed by incubation at 37 °C for 5 min. The remaining surface staining was stripped off and cells were harvested for intracellular fluorescence detection by FACS. Internalized MuSK was quantified. The control sample denotes GFP-positive cells presenting a streptavidin signal. The dimethylsulfoxide sample represents streptavidin-positive cells treated with the solvent dimethylsulfoxide only. Error bars indicate the SEM ( n = 3). (D) COS-7 cells were transiently transfected with SBP-MuSK and GFP or Lrp4-GFP, respectively. MuSK internalization was detected as described in (A). Error bars indicate the SEM (n = 3).

Techniques Used: Activation Assay, Transfection, Incubation, Staining, Fluorescence, FACS

MuSK internalization proceeds via a clathrin-dependent pathway. (A) To determine whether surface MuSK internalizes via clathrin-or caveolin-positive routes, COS-7 cells were transiently transfected with SBP-MuSK. Surface MuSK was stained with Cy3-conjugated streptavidin (red) at 4 °C followed by incubation at 37 °C for 5 min. Endogenous clathrin and caveolin were visualized by antibody staining. MuSK partially colocalizes with these markers (arrowheads in insets). Scale bar = 25 μm. (B) Quantification of MuSK/clathrin, MuSK/caveolin and MuSK/transferrin colocalization using a threshold-and object-based colocalization analysis (as described in the Materials and methods). Colocalization of MuSK with clathrin and caveolin was analyzed at 0 and 5 min of endocytosis. Transferrin and MuSK colocalization was analyzed at 5 min of endocytosis. The peroxisomal marker PTS2-GFP was used as a negative control. Error bars indicate the SEM ( n ≥ 19; from at least two independent experiments). (C) To determine whether dynamin or clathrin are involved in MuSK internalization, COS-7 cells were either treated with the dynamin specific blocker dynasore or co-transfected with GFP-tagged Dyn2K44A or Myc-tagged AP180C. Surface MuSK was stained with streptavidin conjugated-DyLight 649 (red) at 4 °C followed by incubation at 37 °C for 30 min and subsequent stripping of the remaining surface MuSK molecules. Scale bar = 25 μm. (D) To quantify the blockage of MuSK internalization, COS-7 cells were transiently transfected with SBP-MuSK alone or, as indicated, together with AP180C, Eps15DIII or Dyn2K44A. Cells treated with dimethylsulfoxide (DMSO) or dynasore, were stained with streptavidin-conjugated DyLight 649 followed by incubation at 37 °C for 5 min. The remaining surface staining was stripped off and cells were harvested for intracellular fluorescence detection by FACS. A quantification of internalized MuSK is shown. The control sample denotes GFP-positive cells presenting a streptavidin signal. The dimethylsulfoxide sample represents streptavidin-positive cells treated with the solvent dimethylsulfoxide only. Error bars indicate the SEM ( n ≥ 5).
Figure Legend Snippet: MuSK internalization proceeds via a clathrin-dependent pathway. (A) To determine whether surface MuSK internalizes via clathrin-or caveolin-positive routes, COS-7 cells were transiently transfected with SBP-MuSK. Surface MuSK was stained with Cy3-conjugated streptavidin (red) at 4 °C followed by incubation at 37 °C for 5 min. Endogenous clathrin and caveolin were visualized by antibody staining. MuSK partially colocalizes with these markers (arrowheads in insets). Scale bar = 25 μm. (B) Quantification of MuSK/clathrin, MuSK/caveolin and MuSK/transferrin colocalization using a threshold-and object-based colocalization analysis (as described in the Materials and methods). Colocalization of MuSK with clathrin and caveolin was analyzed at 0 and 5 min of endocytosis. Transferrin and MuSK colocalization was analyzed at 5 min of endocytosis. The peroxisomal marker PTS2-GFP was used as a negative control. Error bars indicate the SEM ( n ≥ 19; from at least two independent experiments). (C) To determine whether dynamin or clathrin are involved in MuSK internalization, COS-7 cells were either treated with the dynamin specific blocker dynasore or co-transfected with GFP-tagged Dyn2K44A or Myc-tagged AP180C. Surface MuSK was stained with streptavidin conjugated-DyLight 649 (red) at 4 °C followed by incubation at 37 °C for 30 min and subsequent stripping of the remaining surface MuSK molecules. Scale bar = 25 μm. (D) To quantify the blockage of MuSK internalization, COS-7 cells were transiently transfected with SBP-MuSK alone or, as indicated, together with AP180C, Eps15DIII or Dyn2K44A. Cells treated with dimethylsulfoxide (DMSO) or dynasore, were stained with streptavidin-conjugated DyLight 649 followed by incubation at 37 °C for 5 min. The remaining surface staining was stripped off and cells were harvested for intracellular fluorescence detection by FACS. A quantification of internalized MuSK is shown. The control sample denotes GFP-positive cells presenting a streptavidin signal. The dimethylsulfoxide sample represents streptavidin-positive cells treated with the solvent dimethylsulfoxide only. Error bars indicate the SEM ( n ≥ 5).

Techniques Used: Transfection, Staining, Incubation, Marker, Negative Control, Stripping Membranes, Fluorescence, FACS

Arf6 modulates agrin-induced AChR clustering. (A) C2 myotubes were stimulated with agrin in the presence of dimethylsulfoxide, cytochalasin D, dynasore, myr-Arf6 or AlF. AChRs were stained with α-BGT and visualized by fluorescence microscopy. Scale bar = 25 μm. (B) Quantification of cluster length and cluster number/100 μm is shown. Error bars indicate the SEM ( n > 50). (C) C2 myotubes were treated with agrin for 60 min in the absence or presence of AIF. AChRs were purified using an α-BGT pull-down followed by immunoblotting using antibodies against phosphotyrosine (pTyr) and AChR β, respectively. Quantification of AChR β phosphorylation is shown. Error bars indicate the SEM ( n = 4).
Figure Legend Snippet: Arf6 modulates agrin-induced AChR clustering. (A) C2 myotubes were stimulated with agrin in the presence of dimethylsulfoxide, cytochalasin D, dynasore, myr-Arf6 or AlF. AChRs were stained with α-BGT and visualized by fluorescence microscopy. Scale bar = 25 μm. (B) Quantification of cluster length and cluster number/100 μm is shown. Error bars indicate the SEM ( n > 50). (C) C2 myotubes were treated with agrin for 60 min in the absence or presence of AIF. AChRs were purified using an α-BGT pull-down followed by immunoblotting using antibodies against phosphotyrosine (pTyr) and AChR β, respectively. Quantification of AChR β phosphorylation is shown. Error bars indicate the SEM ( n = 4).

Techniques Used: Staining, Fluorescence, Microscopy, Purification

14) Product Images from "Dynamin- and Lipid Raft-Dependent Entry of Decay-Accelerating Factor (DAF)-Binding and Non-DAF-Binding Coxsackieviruses into Nonpolarized Cells ▿"

Article Title: Dynamin- and Lipid Raft-Dependent Entry of Decay-Accelerating Factor (DAF)-Binding and Non-DAF-Binding Coxsackieviruses into Nonpolarized Cells ▿

Journal:

doi: 10.1128/JVI.01016-09

CVB3-RD entry requires dynamin. CVB3-RD particles were bound to and allowed to enter cells pretreated with DMSO or 200 μM dynasore. Unbound virus was washed off, complete medium with NuSerum and dynasore was added, and virus was allowed to enter
Figure Legend Snippet: CVB3-RD entry requires dynamin. CVB3-RD particles were bound to and allowed to enter cells pretreated with DMSO or 200 μM dynasore. Unbound virus was washed off, complete medium with NuSerum and dynasore was added, and virus was allowed to enter

Techniques Used:

15) Product Images from "Endocytosis, Cytotoxicity, and Translocation of Shiga Toxin-2 Are Stimulated by Infection of Human Intestinal (HCT-8) Monolayers With an Hypervirulent E. coli O157:H7 Lacking stx2 Gene"

Article Title: Endocytosis, Cytotoxicity, and Translocation of Shiga Toxin-2 Are Stimulated by Infection of Human Intestinal (HCT-8) Monolayers With an Hypervirulent E. coli O157:H7 Lacking stx2 Gene

Journal: Frontiers in Cellular and Infection Microbiology

doi: 10.3389/fcimb.2019.00396

Stx2 uptake by HCT-8 cells. (A) Cells pre-treated or not with endocytosis inhibitors were exposed to 100 ng/ml Stx2 and then analyzed by flow cytometry. Histograms represent the log fluorescence of Stx2 for each treatment. A Representative experiment is shown. Amiloride and Dynasore curves are not shown due to superimposition with Stx2 curve. (B) Bar graph representing the Median Intensity of Fluorescence (% MIF) for each inhibitor treatment relative to cells only treated with Stx2. (C) Stx2 positive events (%) for each inhibitor treatment relative to cells only treated with Stx2. Bars represent the mean ± SEM of three independent experiments. * p
Figure Legend Snippet: Stx2 uptake by HCT-8 cells. (A) Cells pre-treated or not with endocytosis inhibitors were exposed to 100 ng/ml Stx2 and then analyzed by flow cytometry. Histograms represent the log fluorescence of Stx2 for each treatment. A Representative experiment is shown. Amiloride and Dynasore curves are not shown due to superimposition with Stx2 curve. (B) Bar graph representing the Median Intensity of Fluorescence (% MIF) for each inhibitor treatment relative to cells only treated with Stx2. (C) Stx2 positive events (%) for each inhibitor treatment relative to cells only treated with Stx2. Bars represent the mean ± SEM of three independent experiments. * p

Techniques Used: Flow Cytometry, Cytometry, Fluorescence

Effect of endocytosis inhibitors on Stx2 cytotoxicity in HCT-8 cells. Cells were pre-incubated with Eliglustat (200 nM, 48 h), MβCD (4 mM, 30 min), Dynasore (80 μM, 30 min), or Amiloride (1 mM, 30 min) and washed twice with PBS before treatment for 4 h of 100 ng/ml Stx2 with or without O157:H7Δstx2. Data is shown as means ± SEM from six independent experiments performed in triplicate. Significant differences were found between the groups, labeled a, b, c, and d.
Figure Legend Snippet: Effect of endocytosis inhibitors on Stx2 cytotoxicity in HCT-8 cells. Cells were pre-incubated with Eliglustat (200 nM, 48 h), MβCD (4 mM, 30 min), Dynasore (80 μM, 30 min), or Amiloride (1 mM, 30 min) and washed twice with PBS before treatment for 4 h of 100 ng/ml Stx2 with or without O157:H7Δstx2. Data is shown as means ± SEM from six independent experiments performed in triplicate. Significant differences were found between the groups, labeled a, b, c, and d.

Techniques Used: Incubation, Labeling

Stx2 translocation in presence of endocytosis inhibitors. Cells were pre-incubated with Eliglustat (200 nM, 48 h), MBCD (4 mM, 30 min), Dynasore (80 μM, 30 min), or Amiloride (1 mM, 30 min), and washed twice with PBS followed by treatment with O157:H7Δstx2 + 100 ng/ml Stx2 for 4 h. Control cells were not pre-treated with inhibitors. (A) Estimated Stx2 concentration in the lower (basolateral) chamber. (B) Relative FITC-Dextran passage (%) to the lower (basolateral) chamber. Significant differences were found between groups with different letters, labeled a, b, and c.
Figure Legend Snippet: Stx2 translocation in presence of endocytosis inhibitors. Cells were pre-incubated with Eliglustat (200 nM, 48 h), MBCD (4 mM, 30 min), Dynasore (80 μM, 30 min), or Amiloride (1 mM, 30 min), and washed twice with PBS followed by treatment with O157:H7Δstx2 + 100 ng/ml Stx2 for 4 h. Control cells were not pre-treated with inhibitors. (A) Estimated Stx2 concentration in the lower (basolateral) chamber. (B) Relative FITC-Dextran passage (%) to the lower (basolateral) chamber. Significant differences were found between groups with different letters, labeled a, b, and c.

Techniques Used: Translocation Assay, Incubation, Concentration Assay, Labeling

16) Product Images from "Inhibitors of endocytosis prevent Wnt/Wingless signalling by reducing the level of basal β-catenin/Armadillo"

Article Title: Inhibitors of endocytosis prevent Wnt/Wingless signalling by reducing the level of basal β-catenin/Armadillo

Journal: Journal of Cell Science

doi: 10.1242/jcs.155424

Endocytosis inhibitors cause a decrease in the level of Armadillo/β-catenin both in stimulated and unstimulated cells. (A) The level of Armadillo increased in S2R + cells that had been treated with Wingless-conditioned medium (lane 2) or SB-216763 (lane 5). This was prevented by treatment with Dyngo-4a (lanes 3 and 6) or Dynasore (lanes 4 and 7). Lamin, Actin and Syntaxin levels were unaffected. (B) Dyngo-4a reversibly reduced the level of Armadillo in uninduced cells. (C) Dyngo-4a reduced signalling-induced accumulation of β-catenin in RKO cells. Cells were pre-incubated with SB-216763 to activate signalling and then exposed to a mixture of SB-216763 and Dyngo-4a. The total time of treatment with either drug is indicated. As in Drosophila cells, Dyngo-4a caused a decrease in β-catenin levels in unstimulated cells. A progressive decrease can be seen after 0.5, 1 and 2 hours of treatment with Dyngo-4a (no SB-216763) in lanes 9–11. (D) The effect of Dyngo-4a and SB-216763 on the level of various components of the Wnt pathway. LRP6, GSK3β and CK1α were largely unaffected, whereas the levels of APC and Axin1 dropped markedly 30–60 minutes after treatment with Dyngo-4a. SB-216763 caused an increase in the amount of β-catenin. This correlated with a decrease in phosphorylated β-catenin (pT42/S37/S33 β-catenin; lane 3), as expected because phosphorylated β-catenin reflects the activity of the degradation complex ( Hernández et al., 2012 ). By contrast, the (mild) decrease in β-catenin caused by Dyngo-4a (lanes 7 and 8) is paralleled by a similar decrease in phosphorylated β-catenin, suggesting that Dyngo-4a impacts on the level of β-catenin through a mechanism that is independent of the destruction complex.
Figure Legend Snippet: Endocytosis inhibitors cause a decrease in the level of Armadillo/β-catenin both in stimulated and unstimulated cells. (A) The level of Armadillo increased in S2R + cells that had been treated with Wingless-conditioned medium (lane 2) or SB-216763 (lane 5). This was prevented by treatment with Dyngo-4a (lanes 3 and 6) or Dynasore (lanes 4 and 7). Lamin, Actin and Syntaxin levels were unaffected. (B) Dyngo-4a reversibly reduced the level of Armadillo in uninduced cells. (C) Dyngo-4a reduced signalling-induced accumulation of β-catenin in RKO cells. Cells were pre-incubated with SB-216763 to activate signalling and then exposed to a mixture of SB-216763 and Dyngo-4a. The total time of treatment with either drug is indicated. As in Drosophila cells, Dyngo-4a caused a decrease in β-catenin levels in unstimulated cells. A progressive decrease can be seen after 0.5, 1 and 2 hours of treatment with Dyngo-4a (no SB-216763) in lanes 9–11. (D) The effect of Dyngo-4a and SB-216763 on the level of various components of the Wnt pathway. LRP6, GSK3β and CK1α were largely unaffected, whereas the levels of APC and Axin1 dropped markedly 30–60 minutes after treatment with Dyngo-4a. SB-216763 caused an increase in the amount of β-catenin. This correlated with a decrease in phosphorylated β-catenin (pT42/S37/S33 β-catenin; lane 3), as expected because phosphorylated β-catenin reflects the activity of the degradation complex ( Hernández et al., 2012 ). By contrast, the (mild) decrease in β-catenin caused by Dyngo-4a (lanes 7 and 8) is paralleled by a similar decrease in phosphorylated β-catenin, suggesting that Dyngo-4a impacts on the level of β-catenin through a mechanism that is independent of the destruction complex.

Techniques Used: Incubation, Activity Assay

17) Product Images from "TLR2 Ligands Induce NF-?B Activation from Endosomal Compartments of Human Monocytes"

Article Title: TLR2 Ligands Induce NF-?B Activation from Endosomal Compartments of Human Monocytes

Journal: PLoS ONE

doi: 10.1371/journal.pone.0080743

Effect of endocytosis inhibitors on TLR2 mediated induction of TNF in primary human monocytes. Monocytes were treated with pharmacological endocytosis inhibitors during 45min prior to treatment for 4h or 24h with LTA (1μg/ml) and Pam 3 CSK 4 (100ng/ml). ( A ) Dose response of the effect of chlorpromazine (CPZ), chloroquine (CHQ) and Dynasore (Dyn) on TNF secretion in TLR2 ligand-activated monocytes. TNF response to LTA and Pam 3 CSK 4 after 24h is strongly reduced by all three endosomal pathway inhibitors. ( B ) Effect of CPZ, CHQ and Dyn on TNF mRNA expression in LTA- and Pam 3 CSK 4 -activated monocytes at 4h. TNF mRNA response to LTA and Pam 3 CSK 4 is reduced by all three endosomal pathway inhibitors. ( C ) Effect of CPZ, CHQ and Dyn on TNF mRNA and TNF production after 4h and 24h, respectively, in IFNγ-activated monocytes. Data were normalized to the TNF production observed in the absence of inhibitors. For all panels data are represented as mean +/- SD of at least 3 independent experiments. *:p ≤ 0.05; ** p ≤ 0.005; *** p ≤ 0.0005.
Figure Legend Snippet: Effect of endocytosis inhibitors on TLR2 mediated induction of TNF in primary human monocytes. Monocytes were treated with pharmacological endocytosis inhibitors during 45min prior to treatment for 4h or 24h with LTA (1μg/ml) and Pam 3 CSK 4 (100ng/ml). ( A ) Dose response of the effect of chlorpromazine (CPZ), chloroquine (CHQ) and Dynasore (Dyn) on TNF secretion in TLR2 ligand-activated monocytes. TNF response to LTA and Pam 3 CSK 4 after 24h is strongly reduced by all three endosomal pathway inhibitors. ( B ) Effect of CPZ, CHQ and Dyn on TNF mRNA expression in LTA- and Pam 3 CSK 4 -activated monocytes at 4h. TNF mRNA response to LTA and Pam 3 CSK 4 is reduced by all three endosomal pathway inhibitors. ( C ) Effect of CPZ, CHQ and Dyn on TNF mRNA and TNF production after 4h and 24h, respectively, in IFNγ-activated monocytes. Data were normalized to the TNF production observed in the absence of inhibitors. For all panels data are represented as mean +/- SD of at least 3 independent experiments. *:p ≤ 0.05; ** p ≤ 0.005; *** p ≤ 0.0005.

Techniques Used: Expressing

18) Product Images from "Clathrin-mediated Endocytosis and Subsequent Endo-Lysosomal Trafficking of Adeno-associated Virus/Phage *"

Article Title: Clathrin-mediated Endocytosis and Subsequent Endo-Lysosomal Trafficking of Adeno-associated Virus/Phage *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M112.369389

Dynamin and Clathrin inhibitors inhibit uptake of RGD-AAVP. A , flow cytometric analysis of uptake of RGD-AAVP ( black bars ) or transferrin control ( white bars ) was performed in HEK293 cells treated with dynasore or ( B ) transiently transfected with GFP-tagged
Figure Legend Snippet: Dynamin and Clathrin inhibitors inhibit uptake of RGD-AAVP. A , flow cytometric analysis of uptake of RGD-AAVP ( black bars ) or transferrin control ( white bars ) was performed in HEK293 cells treated with dynasore or ( B ) transiently transfected with GFP-tagged

Techniques Used: Flow Cytometry, Transfection

19) Product Images from "Human Immunodeficiency Virus type 1 Endocytic Trafficking Through Macrophage Bridging Conduits Facilitates Spread of Infection"

Article Title: Human Immunodeficiency Virus type 1 Endocytic Trafficking Through Macrophage Bridging Conduits Facilitates Spread of Infection

Journal: Journal of Neuroimmune Pharmacology

doi: 10.1007/s11481-011-9298-z

Pathways of macrophage intra- and intercellular HIV-1 trafficking. HIV-1 receptor-mediated entry is regulated by clathrin-coated pits (Miyauchi et al. 2009 ). Upon inhibition of dynamin by dynasore, viral entry is blocked. Clathrin-coated vesicles containing HIV-1 may undergo Rab5/EEA1-dependant fusion with the early sorting endosome(Bucci et al. 1992 ; Zerial and McBride 2001 ). Endosomes can pinch off the vesiculo-tubular network also termed early sorting endosome (Maxfield and McGraw 2004 ) and undergo the following downstream sorting routes: a fuse with the endocytic recycling compartment (ERC; perinuclear region) and then undergo Tfn-like Rab11-mediated recycling to the BC and plasma membrane (Maxfield and McGraw 2004 ). Disruption of this processes by brefeldin A results in accumulation of HIV-1 constituents in large cytosolic compartments (Wang et al. 2001 ). b undergo sorting to the MVB for ILV biogenesis, virus assembly and budding regulated by ESCRT family (Babst et al. 2002 ; Garrus et al. 2001 ; Gill et al. 2007 ). MVB in turn may undergo fusion with Rab11 endosomes to be transported either to the plasma membrane for exosomal release (Simons and Raposo 2009 ) or intercellular transfer through the conduits. Disruption of MVB biogenesis by wortmannin results in agregation of HIV-1 constituents in large vaccuoles (Gruenberg and Stenmark 2004 ). Lysosomes may undergo backfusion with MBV and Rab11 compartments to be targeted at the plasma membrane (secretory non-degrading lysosomes; (Blott and Griffiths 2002 ; Luzio et al. 2005 ) or the bridging conduits. In parallel to endocytic entry and intercellular trafficking, virion-plasma membrane fusion may also occur followed by uncoating, reverse trancription, formation of pre-integration complexes (PIC), and RNA expression (Brass et al. 2008 ). Thick blue arrows indicate trafficking routes that target HIV-1 directly to the conduits
Figure Legend Snippet: Pathways of macrophage intra- and intercellular HIV-1 trafficking. HIV-1 receptor-mediated entry is regulated by clathrin-coated pits (Miyauchi et al. 2009 ). Upon inhibition of dynamin by dynasore, viral entry is blocked. Clathrin-coated vesicles containing HIV-1 may undergo Rab5/EEA1-dependant fusion with the early sorting endosome(Bucci et al. 1992 ; Zerial and McBride 2001 ). Endosomes can pinch off the vesiculo-tubular network also termed early sorting endosome (Maxfield and McGraw 2004 ) and undergo the following downstream sorting routes: a fuse with the endocytic recycling compartment (ERC; perinuclear region) and then undergo Tfn-like Rab11-mediated recycling to the BC and plasma membrane (Maxfield and McGraw 2004 ). Disruption of this processes by brefeldin A results in accumulation of HIV-1 constituents in large cytosolic compartments (Wang et al. 2001 ). b undergo sorting to the MVB for ILV biogenesis, virus assembly and budding regulated by ESCRT family (Babst et al. 2002 ; Garrus et al. 2001 ; Gill et al. 2007 ). MVB in turn may undergo fusion with Rab11 endosomes to be transported either to the plasma membrane for exosomal release (Simons and Raposo 2009 ) or intercellular transfer through the conduits. Disruption of MVB biogenesis by wortmannin results in agregation of HIV-1 constituents in large vaccuoles (Gruenberg and Stenmark 2004 ). Lysosomes may undergo backfusion with MBV and Rab11 compartments to be targeted at the plasma membrane (secretory non-degrading lysosomes; (Blott and Griffiths 2002 ; Luzio et al. 2005 ) or the bridging conduits. In parallel to endocytic entry and intercellular trafficking, virion-plasma membrane fusion may also occur followed by uncoating, reverse trancription, formation of pre-integration complexes (PIC), and RNA expression (Brass et al. 2008 ). Thick blue arrows indicate trafficking routes that target HIV-1 directly to the conduits

Techniques Used: Inhibition, RNA Expression

20) Product Images from "Alzheimer’s disease pathology propagation by exosomes containing toxic amyloid-beta oligomers"

Article Title: Alzheimer’s disease pathology propagation by exosomes containing toxic amyloid-beta oligomers

Journal: Acta Neuropathologica

doi: 10.1007/s00401-018-1868-1

The uptake of exosomes and the subsequent spreading of oAß is dynamin-dependent. a , b Uptake of PKH67 labelled exosomes or oAβ-AF700 in dSH-SY5Y cells. Cells were pre-incubated with the indicated inhibitors for 30 min, then exposed to exosomes or oAβ-AF700. After 3 h incubation, samples were collected and the proportion of cells with uptake was quantified by flow cytometry and related to untreated control (dotted line). c Flow cytometry analysis of oAβ transfer in presence of dynasore in coverslip and transwell co-culture models. After dynasore treatment there is a significant decrease of the proportion of cells with oAβ transfer in both models (control, dotted line). d Transfer of exosomes isolated from oAβ treated cells causes cytotoxicity in recipient cells compared to untreated control as shown by LDH assay, whereas dynasore treatment significantly reduces the cytotoxic effect versus untreated control (dotted line). Data are represented as the mean ± SEM, NS, not significant; n = 4; ** p
Figure Legend Snippet: The uptake of exosomes and the subsequent spreading of oAß is dynamin-dependent. a , b Uptake of PKH67 labelled exosomes or oAβ-AF700 in dSH-SY5Y cells. Cells were pre-incubated with the indicated inhibitors for 30 min, then exposed to exosomes or oAβ-AF700. After 3 h incubation, samples were collected and the proportion of cells with uptake was quantified by flow cytometry and related to untreated control (dotted line). c Flow cytometry analysis of oAβ transfer in presence of dynasore in coverslip and transwell co-culture models. After dynasore treatment there is a significant decrease of the proportion of cells with oAβ transfer in both models (control, dotted line). d Transfer of exosomes isolated from oAβ treated cells causes cytotoxicity in recipient cells compared to untreated control as shown by LDH assay, whereas dynasore treatment significantly reduces the cytotoxic effect versus untreated control (dotted line). Data are represented as the mean ± SEM, NS, not significant; n = 4; ** p

Techniques Used: Incubation, Flow Cytometry, Cytometry, Co-Culture Assay, Isolation, Lactate Dehydrogenase Assay

21) Product Images from "Endocytosis of Murine Norovirus 1 into Murine Macrophages Is Dependent on Dynamin II and Cholesterol ▿"

Article Title: Endocytosis of Murine Norovirus 1 into Murine Macrophages Is Dependent on Dynamin II and Cholesterol ▿

Journal: Journal of Virology

doi: 10.1128/JVI.00331-10

MNV-1 infection requires dynamin II. RAW 264.7 cells (A) and BMDMs (B) were pretreated with dynasore at the indicated concentration for 30 min, infected with MNV-1 or VSV on ice for 1 h, and washed with PBS. At 8 h (RAW 264.7 cells) or 10 h (BMDMs) postinfection,
Figure Legend Snippet: MNV-1 infection requires dynamin II. RAW 264.7 cells (A) and BMDMs (B) were pretreated with dynasore at the indicated concentration for 30 min, infected with MNV-1 or VSV on ice for 1 h, and washed with PBS. At 8 h (RAW 264.7 cells) or 10 h (BMDMs) postinfection,

Techniques Used: Infection, Concentration Assay

22) Product Images from "Alzheimer’s disease pathology propagation by exosomes containing toxic amyloid-beta oligomers"

Article Title: Alzheimer’s disease pathology propagation by exosomes containing toxic amyloid-beta oligomers

Journal: Acta Neuropathologica

doi: 10.1007/s00401-018-1868-1

The uptake of exosomes and the subsequent spreading of oAß is dynamin-dependent. a , b Uptake of PKH67 labelled exosomes or oAβ-AF700 in dSH-SY5Y cells. Cells were pre-incubated with the indicated inhibitors for 30 min, then exposed to exosomes or oAβ-AF700. After 3 h incubation, samples were collected and the proportion of cells with uptake was quantified by flow cytometry and related to untreated control (dotted line). c Flow cytometry analysis of oAβ transfer in presence of dynasore in coverslip and transwell co-culture models. After dynasore treatment there is a significant decrease of the proportion of cells with oAβ transfer in both models (control, dotted line). d Transfer of exosomes isolated from oAβ treated cells causes cytotoxicity in recipient cells compared to untreated control as shown by LDH assay, whereas dynasore treatment significantly reduces the cytotoxic effect versus untreated control (dotted line). Data are represented as the mean ± SEM, NS, not significant; n = 4; ** p
Figure Legend Snippet: The uptake of exosomes and the subsequent spreading of oAß is dynamin-dependent. a , b Uptake of PKH67 labelled exosomes or oAβ-AF700 in dSH-SY5Y cells. Cells were pre-incubated with the indicated inhibitors for 30 min, then exposed to exosomes or oAβ-AF700. After 3 h incubation, samples were collected and the proportion of cells with uptake was quantified by flow cytometry and related to untreated control (dotted line). c Flow cytometry analysis of oAβ transfer in presence of dynasore in coverslip and transwell co-culture models. After dynasore treatment there is a significant decrease of the proportion of cells with oAβ transfer in both models (control, dotted line). d Transfer of exosomes isolated from oAβ treated cells causes cytotoxicity in recipient cells compared to untreated control as shown by LDH assay, whereas dynasore treatment significantly reduces the cytotoxic effect versus untreated control (dotted line). Data are represented as the mean ± SEM, NS, not significant; n = 4; ** p

Techniques Used: Incubation, Flow Cytometry, Cytometry, Co-Culture Assay, Isolation, Lactate Dehydrogenase Assay

23) Product Images from "Caveolin-1 and Dynamin-2 Are Essential for Removal of the Complement C5b-9 Complex via Endocytosis *"

Article Title: Caveolin-1 and Dynamin-2 Are Essential for Removal of the Complement C5b-9 Complex via Endocytosis *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M111.333039

Dynamin protects K562 cells from complement-dependent cytotoxicity. K562 cells were transfected with the K44A plasmid or an empty vector control (EGFP) for 24, 48 or 72 h ( A ) or for 30 min at 37 °C with Dynasore at different concentrations ( B
Figure Legend Snippet: Dynamin protects K562 cells from complement-dependent cytotoxicity. K562 cells were transfected with the K44A plasmid or an empty vector control (EGFP) for 24, 48 or 72 h ( A ) or for 30 min at 37 °C with Dynasore at different concentrations ( B

Techniques Used: Transfection, Plasmid Preparation

24) Product Images from "Neurotransmitter-Triggered Transfer of Exosomes Mediates Oligodendrocyte-Neuron Communication"

Article Title: Neurotransmitter-Triggered Transfer of Exosomes Mediates Oligodendrocyte-Neuron Communication

Journal: PLoS Biology

doi: 10.1371/journal.pbio.1001604

Primary cortical neurons internalize oligodendroglial exosomes. (A–D) pOL were stained with the lipophilic dye PKH67 (green), washed, and subsequently co-cultured in Boyden chambers for 2 d with mixed neural cultures containing astrocytes (A, B, blue marker GFAP), oligodendrocytes (B, red marker O4), and microglia (A, red marker F4/80, B, arrowheads) or with CN (C, red marker Tuj1). Scale bar, 20 µm. (D) Quantification of exosome uptake by the different types of target cells. Error bars, SEM, ( n = 3). (E) Fluorescent exosomes containing SIRT2-EYFP and PLP-EGFP were purified by sucrose density gradient centrifugation from Oli-neu cells and co-incubated with CN for 24 h. The Western blot depicts EGFP and the exosomal marker Tsg101 in gradient fractions. Images show maximum projections of confocal Z-stacks of Tuj1-stained neurons after incubation with exosomes (scale bar, 5 µm). (F) Western blots of purified Oli-neu exosomes (input, left lane) and neuronal lysates after treatment with exosomes (Exo). EGFP/EYFP depicts exosome markers, Tubulin (Tub) is used as normalization standard. Relative exosome uptake reflects normalized signals of SIRT2-EYFP and PLP-EGFP associated with neuronal lysates ( n = 8). (G) To remove surface-bound exosomes, neurons were treated with trypsin (Tryp) before lysis ( n = 5). (H–K) Boyden chamber co-culture of oligodendroglial cells and CN for 2–3 d and analysis of exosomal PLP and SIRT2 in neurons by Western blot (H, I, and K) or immunostaining (J). (H) PLP-EGFP and SIRT2-EYFP expressing Oli-neu cells were treated or not with 5 µM GW4869 inhibiting exosome release ( n = 6). (I) pOL were treated with 100 µM glutamate stimulating exosome release ( n = 5). (J) Co-culture of CN with PKH67-labelled (green) pOL and immunostaining of CN with Tuj1 (red) and the late endosomal/lysosomal marker LAMP1 (blue). Maximum projection of a confocal Z-stack. Scale bar, 5 µm. (K) Neurons were pre-treated with Dynasore and co-cultured for 1 d with Oli-neu cells releasing SIRT2-EYFP and PLP-EGFP labeled exosomes ( n = 4). Error bars, SEM (* p
Figure Legend Snippet: Primary cortical neurons internalize oligodendroglial exosomes. (A–D) pOL were stained with the lipophilic dye PKH67 (green), washed, and subsequently co-cultured in Boyden chambers for 2 d with mixed neural cultures containing astrocytes (A, B, blue marker GFAP), oligodendrocytes (B, red marker O4), and microglia (A, red marker F4/80, B, arrowheads) or with CN (C, red marker Tuj1). Scale bar, 20 µm. (D) Quantification of exosome uptake by the different types of target cells. Error bars, SEM, ( n = 3). (E) Fluorescent exosomes containing SIRT2-EYFP and PLP-EGFP were purified by sucrose density gradient centrifugation from Oli-neu cells and co-incubated with CN for 24 h. The Western blot depicts EGFP and the exosomal marker Tsg101 in gradient fractions. Images show maximum projections of confocal Z-stacks of Tuj1-stained neurons after incubation with exosomes (scale bar, 5 µm). (F) Western blots of purified Oli-neu exosomes (input, left lane) and neuronal lysates after treatment with exosomes (Exo). EGFP/EYFP depicts exosome markers, Tubulin (Tub) is used as normalization standard. Relative exosome uptake reflects normalized signals of SIRT2-EYFP and PLP-EGFP associated with neuronal lysates ( n = 8). (G) To remove surface-bound exosomes, neurons were treated with trypsin (Tryp) before lysis ( n = 5). (H–K) Boyden chamber co-culture of oligodendroglial cells and CN for 2–3 d and analysis of exosomal PLP and SIRT2 in neurons by Western blot (H, I, and K) or immunostaining (J). (H) PLP-EGFP and SIRT2-EYFP expressing Oli-neu cells were treated or not with 5 µM GW4869 inhibiting exosome release ( n = 6). (I) pOL were treated with 100 µM glutamate stimulating exosome release ( n = 5). (J) Co-culture of CN with PKH67-labelled (green) pOL and immunostaining of CN with Tuj1 (red) and the late endosomal/lysosomal marker LAMP1 (blue). Maximum projection of a confocal Z-stack. Scale bar, 5 µm. (K) Neurons were pre-treated with Dynasore and co-cultured for 1 d with Oli-neu cells releasing SIRT2-EYFP and PLP-EGFP labeled exosomes ( n = 4). Error bars, SEM (* p

Techniques Used: Staining, Cell Culture, Marker, Plasmid Purification, Purification, Gradient Centrifugation, Incubation, Western Blot, Lysis, Co-Culture Assay, Immunostaining, Expressing, Labeling

25) Product Images from "Clathrin-Independent Trafficking of AMPA Receptors"

Article Title: Clathrin-Independent Trafficking of AMPA Receptors

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.3571-14.2015

CME is required for NMDAR–LTD but not for basal synaptic transmission or synaptic downscaling. A , Treatment with dynasore does not affect basal excitatory synaptic transmission. Time course of EPSC amplitude evoked at 0.2 Hz at Schaffer collateral–CA1
Figure Legend Snippet: CME is required for NMDAR–LTD but not for basal synaptic transmission or synaptic downscaling. A , Treatment with dynasore does not affect basal excitatory synaptic transmission. Time course of EPSC amplitude evoked at 0.2 Hz at Schaffer collateral–CA1

Techniques Used: Transmission Assay

26) Product Images from "Specific Aptamer-Based Probe for Analyzing Biomarker MCP Entry Into Singapore Grouper Iridovirus-Infected Host Cells via Clathrin-Mediated Endocytosis"

Article Title: Specific Aptamer-Based Probe for Analyzing Biomarker MCP Entry Into Singapore Grouper Iridovirus-Infected Host Cells via Clathrin-Mediated Endocytosis

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2020.01206

MCP endocytosis depends on dynamin. Dynamin is essential for clathrin-coated vesicle formation and membrane budding in the late stage, and dynasore is a specific inhibitor of dynamin. (A) The fluorescent Cy5 signal inside the SGIV-infected cells was significantly reduced in the dynasore-treated cells. (B) Entry efficiency of the Q5-MCP complex was greatly and dose-dependently reduced in the presence of dynasore (to 75.4% with 10 μM dynasore, to 55.9% at 20 μM dynasore). p
Figure Legend Snippet: MCP endocytosis depends on dynamin. Dynamin is essential for clathrin-coated vesicle formation and membrane budding in the late stage, and dynasore is a specific inhibitor of dynamin. (A) The fluorescent Cy5 signal inside the SGIV-infected cells was significantly reduced in the dynasore-treated cells. (B) Entry efficiency of the Q5-MCP complex was greatly and dose-dependently reduced in the presence of dynasore (to 75.4% with 10 μM dynasore, to 55.9% at 20 μM dynasore). p

Techniques Used: Infection

27) Product Images from "A novel function of FAF1, which induces dopaminergic neuronal death through cell-to-cell transmission"

Article Title: A novel function of FAF1, which induces dopaminergic neuronal death through cell-to-cell transmission

Journal: Cell Communication and Signaling : CCS

doi: 10.1186/s12964-020-00632-8

Transferred FAF1 in adjacent cells maintains its death function. Donor cells were transfected with VC or 3xFlag-FAF1 plasmid. At 24 h after transfection, the culture medium was replaced with serum-free medium, and the cells were cultured for 24 h. a The CM was applied to recipient cells for 48 h. Heat-inactivated CM was boiled for 10 min. Cell death was determined by measuring PI uptake using a flow cytometer. b Rat donor primary neuronal cells were transduced with AAV1-hFAF1 viral vectors. At 3 days after transduction, the culture medium was replaced with serum-free neurobasal medium for 48 h. The CM was applied to recipient primary neuronal cells for 48 h. Cell death was determined by measuring PI uptake using a flow cytometer. c The CM from vector-transfected cells or FAF1-transfected cells containing vehicle (DMSO), z-VAD-fmk (100 μM), DPQ (30 μM), or Nec-1 (50 μM) for 48 h was applied to recipient cells. d After the CM was fractionated into the retained fraction (exosomal FAF1) and flow-through (vesicle-free FAF1), each CM fraction was applied to recipient cells for 48 h. e FAF1 was immunodepleted from CM with anti-FAF1 monoclonal antibody, and the CM was applied to recipient cells for 48 h. f After the recipient cells were pretreated with Dynasore (80 μM), heparin (200 μg/ml), or heparinase III (0.01 IU/ml) for 24 h, CM was applied to recipient cells for 48 h. Cell death was determined by measuring PI uptake using a flow cytometer. Data are expressed as the mean ± S.D. of three independent experiments ( n = 3). Statistical comparisons were performed using ANOVA followed by Tukey’s HSD post hoc analysis. ** P
Figure Legend Snippet: Transferred FAF1 in adjacent cells maintains its death function. Donor cells were transfected with VC or 3xFlag-FAF1 plasmid. At 24 h after transfection, the culture medium was replaced with serum-free medium, and the cells were cultured for 24 h. a The CM was applied to recipient cells for 48 h. Heat-inactivated CM was boiled for 10 min. Cell death was determined by measuring PI uptake using a flow cytometer. b Rat donor primary neuronal cells were transduced with AAV1-hFAF1 viral vectors. At 3 days after transduction, the culture medium was replaced with serum-free neurobasal medium for 48 h. The CM was applied to recipient primary neuronal cells for 48 h. Cell death was determined by measuring PI uptake using a flow cytometer. c The CM from vector-transfected cells or FAF1-transfected cells containing vehicle (DMSO), z-VAD-fmk (100 μM), DPQ (30 μM), or Nec-1 (50 μM) for 48 h was applied to recipient cells. d After the CM was fractionated into the retained fraction (exosomal FAF1) and flow-through (vesicle-free FAF1), each CM fraction was applied to recipient cells for 48 h. e FAF1 was immunodepleted from CM with anti-FAF1 monoclonal antibody, and the CM was applied to recipient cells for 48 h. f After the recipient cells were pretreated with Dynasore (80 μM), heparin (200 μg/ml), or heparinase III (0.01 IU/ml) for 24 h, CM was applied to recipient cells for 48 h. Cell death was determined by measuring PI uptake using a flow cytometer. Data are expressed as the mean ± S.D. of three independent experiments ( n = 3). Statistical comparisons were performed using ANOVA followed by Tukey’s HSD post hoc analysis. ** P

Techniques Used: Transfection, Plasmid Preparation, Cell Culture, Flow Cytometry, Transduction

28) Product Images from "Cisplatin induces the release of extracellular vesicles from ovarian cancer cells that can induce invasiveness and drug resistance in bystander cells"

Article Title: Cisplatin induces the release of extracellular vesicles from ovarian cancer cells that can induce invasiveness and drug resistance in bystander cells

Journal: Philosophical Transactions of the Royal Society B: Biological Sciences

doi: 10.1098/rstb.2017.0065

EV inhibitors alter the cisplatin sensitivity of ovarian cancer cell lines. Cells were seeded in 96-well plates (day 0); they were pre-treated on day 2 for 30 min with heparin ( a – c ), amiloride or dynasore ( d ). They were then treated with varying concentrations of cisplatin for 3 h. Viability was quantified by the MTT assay after 48 h. There was a significant increase in sensitivity to cisplatin in A2780 ( a ) ( p
Figure Legend Snippet: EV inhibitors alter the cisplatin sensitivity of ovarian cancer cell lines. Cells were seeded in 96-well plates (day 0); they were pre-treated on day 2 for 30 min with heparin ( a – c ), amiloride or dynasore ( d ). They were then treated with varying concentrations of cisplatin for 3 h. Viability was quantified by the MTT assay after 48 h. There was a significant increase in sensitivity to cisplatin in A2780 ( a ) ( p

Techniques Used: MTT Assay

29) Product Images from "Phosphatidylinositol 3-Kinase Class II α-Isoform PI3K-C2α Is Required for Transforming Growth Factor β-induced Smad Signaling in Endothelial Cells *"

Article Title: Phosphatidylinositol 3-Kinase Class II α-Isoform PI3K-C2α Is Required for Transforming Growth Factor β-induced Smad Signaling in Endothelial Cells *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.601484

PI3K-C2 α is required for TGF β 1-induced internalization of TGF β receptor into the early endosomes in EC. A , effects of C2α depletion or dynasore on TGFβ1-induced internalization of endogenous ALK5. The cells were
Figure Legend Snippet: PI3K-C2 α is required for TGF β 1-induced internalization of TGF β receptor into the early endosomes in EC. A , effects of C2α depletion or dynasore on TGFβ1-induced internalization of endogenous ALK5. The cells were

Techniques Used:

Receptor endocytosis and ALK5 are necessary for TGF β 1-induced angiogenesis in vivo . Matrigels containing of PBS ( vehicle ) or TGFβ1 and dynasore (200 μ m ) or iALK5 (20 μ m ) were injected into the subcutaneous tissues on the
Figure Legend Snippet: Receptor endocytosis and ALK5 are necessary for TGF β 1-induced angiogenesis in vivo . Matrigels containing of PBS ( vehicle ) or TGFβ1 and dynasore (200 μ m ) or iALK5 (20 μ m ) were injected into the subcutaneous tissues on the

Techniques Used: In Vivo, Injection

30) Product Images from "Antiviral effects of ferric ammonium citrate"

Article Title: Antiviral effects of ferric ammonium citrate

Journal: Cell Discovery

doi: 10.1038/s41421-018-0013-6

FAC-induced intracellular vesicle fusion. a PR8-infected A549 cells were treated in the presence or absence of FAC for 6 h or 12 h. Cells were analyzed under confocal microscopy for PR8 NP protein. Scale bars, 20 μm. b PR8-infected Hela cells were treated in the presence or absence of FAC for 6 h. Cells were analyzed under confocal microscopy for PR8 NP protein and RAB7. Scale bars, 10 μm. c A549 cells were infected with PR8 ± FAC for 6 h and analyzed via electron microscopy. Endosomes (irregular circles, white inside) and endolysosomes (irregular circles, dark inside) were marked by white lines. N nucleus. M mitochondrion. d Hela cells were treated with or without FAC for 6 h and analyzed through confocal microscopy for RAB7. Average RAB7-positive vesicle area and total RAB7 positive vesicle area per cell were statistically analyzed. Scale bars, 20 μm. e Hela cells were treated with Dynasore (Dyn) and/or FAC for indicated time. Cells were stained with 0.1% crystal violet and analyzed under bright field microscopy. Scale bars, 20 μm. Data are representative of three independent experiments. Error bars represent SD
Figure Legend Snippet: FAC-induced intracellular vesicle fusion. a PR8-infected A549 cells were treated in the presence or absence of FAC for 6 h or 12 h. Cells were analyzed under confocal microscopy for PR8 NP protein. Scale bars, 20 μm. b PR8-infected Hela cells were treated in the presence or absence of FAC for 6 h. Cells were analyzed under confocal microscopy for PR8 NP protein and RAB7. Scale bars, 10 μm. c A549 cells were infected with PR8 ± FAC for 6 h and analyzed via electron microscopy. Endosomes (irregular circles, white inside) and endolysosomes (irregular circles, dark inside) were marked by white lines. N nucleus. M mitochondrion. d Hela cells were treated with or without FAC for 6 h and analyzed through confocal microscopy for RAB7. Average RAB7-positive vesicle area and total RAB7 positive vesicle area per cell were statistically analyzed. Scale bars, 20 μm. e Hela cells were treated with Dynasore (Dyn) and/or FAC for indicated time. Cells were stained with 0.1% crystal violet and analyzed under bright field microscopy. Scale bars, 20 μm. Data are representative of three independent experiments. Error bars represent SD

Techniques Used: Infection, Confocal Microscopy, Electron Microscopy, Staining, Microscopy

31) Product Images from "Altering integrin engagement regulates membrane localization of Kir2.1 channels"

Article Title: Altering integrin engagement regulates membrane localization of Kir2.1 channels

Journal: Journal of Cell Science

doi: 10.1242/jcs.225383

Effects of microtubular trafficking and endocytosis on FAKi-induced I K1 reduction in micropatterned Ex293 cells. (A) Representative images of micropatterned Ex293 cells immunostained for active β1-integrin after exposure to 12 µM Nocodazole (Noco, top) or 100 µM FAK-inhibitor (FAKi)+12 µM Nocodazole (Noco+FAKi, bottom) for 2, 4 or 6 h. ‘VC’ represents a 6-h exposure to vehicle control. (B,C) Corresponding quantifications of active β1-integrin coverage (B, n =10 cells per group; mean±s.e.m.) and I K1 density measured at −90 mV (C, n =5–14 cells per group; mean±s.e.m.). (D) Representative images of Ex293 cells stained for active β1-integrin after exposure to 25 µM Dynasore (Dyn, top) or 100 µM FAK-inhibitor (FAKi)+25 µM Dynasore (Dyn+FAKi, bottom) for 2, 4 or 6 h. (E,F) Corresponding quantifications of active β1-integrin coverage (E, n =10 cells cells per group; mean±s.e.m. ) and I K1 density measured at −90 mV (F, n =6–20 cells per group; mean±s.e.m.). (G) Correlation between I K1 density and active β1-integrin coverage for different interventions. (H) Representative images of Ex293 cells stained for vinculin after exposure to 25 µM Dynasore (Dyn, top) or 100 µM FAK-inhibitor (FAKi)+25 µM Dynasore (Dyn+FAKi, bottom) for 2, 4 or 6 h. (I) Corresponding quantifications of total FA coverage in Ex293 cells ( n =6–13 cells per group; mean±s.e.m.). * P
Figure Legend Snippet: Effects of microtubular trafficking and endocytosis on FAKi-induced I K1 reduction in micropatterned Ex293 cells. (A) Representative images of micropatterned Ex293 cells immunostained for active β1-integrin after exposure to 12 µM Nocodazole (Noco, top) or 100 µM FAK-inhibitor (FAKi)+12 µM Nocodazole (Noco+FAKi, bottom) for 2, 4 or 6 h. ‘VC’ represents a 6-h exposure to vehicle control. (B,C) Corresponding quantifications of active β1-integrin coverage (B, n =10 cells per group; mean±s.e.m.) and I K1 density measured at −90 mV (C, n =5–14 cells per group; mean±s.e.m.). (D) Representative images of Ex293 cells stained for active β1-integrin after exposure to 25 µM Dynasore (Dyn, top) or 100 µM FAK-inhibitor (FAKi)+25 µM Dynasore (Dyn+FAKi, bottom) for 2, 4 or 6 h. (E,F) Corresponding quantifications of active β1-integrin coverage (E, n =10 cells cells per group; mean±s.e.m. ) and I K1 density measured at −90 mV (F, n =6–20 cells per group; mean±s.e.m.). (G) Correlation between I K1 density and active β1-integrin coverage for different interventions. (H) Representative images of Ex293 cells stained for vinculin after exposure to 25 µM Dynasore (Dyn, top) or 100 µM FAK-inhibitor (FAKi)+25 µM Dynasore (Dyn+FAKi, bottom) for 2, 4 or 6 h. (I) Corresponding quantifications of total FA coverage in Ex293 cells ( n =6–13 cells per group; mean±s.e.m.). * P

Techniques Used: Staining

Insertion and internalization of K ir 2.1 into the membrane of micropatterned HEK293 cells. (A) Representative snapshots of bleach-corrected K ir 2.1–tdTomato live fluorescence in micropatterned HEK293 cells shown pre-bleaching and at various times post-bleaching. White boxes indicate edge and corner regions used for FRAP analysis. (B) Experimental data and single-exponential fits of K ir 2.1–tdTomato fluorescence recovery in the corner versus edge regions. (C) Corresponding quantifications of time constant of recovery (τ, top) and mobile fraction ( M f , bottom n =19 cells per group; mean±s.e.m.). (D) Representative snapshots of bleach-corrected K ir 2.1–tdTomato live fluorescence in micropatterned HEK293 cells treated with 25 µM dynasore for 4 h shown pre-bleaching and at various times post-bleaching. Boxes indicate edge and corner regions used for FRAP analysis. (E) Experimental data and single-exponential fits of K ir 2.1–tdTomato fluorescence recovery in the corner versus edge regions. (F) Corresponding quantifications of time constant of recovery (τ, top) and mobile fraction ( M f , bottom, n =17 cells per group; mean±s.e.m.). * P
Figure Legend Snippet: Insertion and internalization of K ir 2.1 into the membrane of micropatterned HEK293 cells. (A) Representative snapshots of bleach-corrected K ir 2.1–tdTomato live fluorescence in micropatterned HEK293 cells shown pre-bleaching and at various times post-bleaching. White boxes indicate edge and corner regions used for FRAP analysis. (B) Experimental data and single-exponential fits of K ir 2.1–tdTomato fluorescence recovery in the corner versus edge regions. (C) Corresponding quantifications of time constant of recovery (τ, top) and mobile fraction ( M f , bottom n =19 cells per group; mean±s.e.m.). (D) Representative snapshots of bleach-corrected K ir 2.1–tdTomato live fluorescence in micropatterned HEK293 cells treated with 25 µM dynasore for 4 h shown pre-bleaching and at various times post-bleaching. Boxes indicate edge and corner regions used for FRAP analysis. (E) Experimental data and single-exponential fits of K ir 2.1–tdTomato fluorescence recovery in the corner versus edge regions. (F) Corresponding quantifications of time constant of recovery (τ, top) and mobile fraction ( M f , bottom, n =17 cells per group; mean±s.e.m.). * P

Techniques Used: Fluorescence

32) Product Images from "Dynamin-2 Stabilizes the HIV-1 Fusion Pore with a Low Oligomeric State"

Article Title: Dynamin-2 Stabilizes the HIV-1 Fusion Pore with a Low Oligomeric State

Journal: Cell Reports

doi: 10.1016/j.celrep.2016.12.032

Cell-Cell Fusion Experiments Show that Dynamin-2 Stabilizes HIV-1 Env Mediated Fusion (A) A cartoon depicting the strategy followed for our cell-cell fusion assays is shown. Briefly, HEK293T cells expressing freely diffusing GFPs and JRFL Env (effector cells) were added onto TZM-bl reporter cells expressing freely diffusing mCherry (target cells) at 4°C for 30 min. Shifting the temperature under the microscope at 37°C permitted the visualization of JRFL Env mediated cell-cell fusion, measured by time-resolved two color confocal fluorescence microscopy. (B and C) Micrographs showing TZM-bl cells transfected with mCherry exposed to HEK293T cells expressing JR-FL and eGFP untreated (B) or treated with 400 μM of dynasore (C). The transmission channel is also included (in gray). Different time points show cells undergoing cell-cell fusion 2 min after changing the temperature to 37°C (B) or 4 min (C). These events are shown with a white arrow. The error bar represents 20 μm. (D) Composite micrographs of a region of interest depicting cell-cell fusion showing transmission and green channel (HEK293T cells expressing GFP and JRFL Env, left column), transmission and red channel (TZM-bl cells expressing mCherry, middle), and merged channels (right column) at two different time lags: 0 min (no fusion) and 6 min (cell-cell fusion completed shown by the concomitant transfer of red mCherry fluorescent proteins from target cells toward effector cells and eGFP fluorescent proteins from effector cells toward target cells). The scale bar represents 10 μm. (E) The fluorescence intensities were recovered as a function of time integrating both signals (red and green) coming from two single events from target cells in the absence of dynasore (left) and in the presence of 400 μM dynasore (middle). The flickering of the fusion pore is only observed in cells treated with dynasore. The cumulative distribution of individual cell-cell fusion events comparing untreated cells (green dots, n = 17) against dynasore treated cells (small red dots, n = 20) is shown in the right image, evidencing a delay of around 3 min for cells treated with DNM2 inhibitor dynasore. (F) HEK293T cells expressing freely diffusing GFPs and VSVG Env (effector cells) were added onto TZM-bl reporter cells expressing freely diffusing mCherry (target cells) at room temperature for 30 min. Shifting the pH using a citrate buffer at pH ∼5 permitted us to visualize VSVG Env mediated cell-cell fusion, measured by time-resolved two color confocal fluorescence microscopy. The left image shows a representative example without dynasore treatment, and the middle image being an example of cell-cell fusion treated with 400 μM dynasore. Flickering of the pore was never observed in this case. The cumulative distribution of individual cell-cell fusion events comparing untreated cells (green dots, n = 16) against dynasore treated cells (small red dots, n = 15) is shown in the right image, evidencing synchronous fusion kinetics.
Figure Legend Snippet: Cell-Cell Fusion Experiments Show that Dynamin-2 Stabilizes HIV-1 Env Mediated Fusion (A) A cartoon depicting the strategy followed for our cell-cell fusion assays is shown. Briefly, HEK293T cells expressing freely diffusing GFPs and JRFL Env (effector cells) were added onto TZM-bl reporter cells expressing freely diffusing mCherry (target cells) at 4°C for 30 min. Shifting the temperature under the microscope at 37°C permitted the visualization of JRFL Env mediated cell-cell fusion, measured by time-resolved two color confocal fluorescence microscopy. (B and C) Micrographs showing TZM-bl cells transfected with mCherry exposed to HEK293T cells expressing JR-FL and eGFP untreated (B) or treated with 400 μM of dynasore (C). The transmission channel is also included (in gray). Different time points show cells undergoing cell-cell fusion 2 min after changing the temperature to 37°C (B) or 4 min (C). These events are shown with a white arrow. The error bar represents 20 μm. (D) Composite micrographs of a region of interest depicting cell-cell fusion showing transmission and green channel (HEK293T cells expressing GFP and JRFL Env, left column), transmission and red channel (TZM-bl cells expressing mCherry, middle), and merged channels (right column) at two different time lags: 0 min (no fusion) and 6 min (cell-cell fusion completed shown by the concomitant transfer of red mCherry fluorescent proteins from target cells toward effector cells and eGFP fluorescent proteins from effector cells toward target cells). The scale bar represents 10 μm. (E) The fluorescence intensities were recovered as a function of time integrating both signals (red and green) coming from two single events from target cells in the absence of dynasore (left) and in the presence of 400 μM dynasore (middle). The flickering of the fusion pore is only observed in cells treated with dynasore. The cumulative distribution of individual cell-cell fusion events comparing untreated cells (green dots, n = 17) against dynasore treated cells (small red dots, n = 20) is shown in the right image, evidencing a delay of around 3 min for cells treated with DNM2 inhibitor dynasore. (F) HEK293T cells expressing freely diffusing GFPs and VSVG Env (effector cells) were added onto TZM-bl reporter cells expressing freely diffusing mCherry (target cells) at room temperature for 30 min. Shifting the pH using a citrate buffer at pH ∼5 permitted us to visualize VSVG Env mediated cell-cell fusion, measured by time-resolved two color confocal fluorescence microscopy. The left image shows a representative example without dynasore treatment, and the middle image being an example of cell-cell fusion treated with 400 μM dynasore. Flickering of the pore was never observed in this case. The cumulative distribution of individual cell-cell fusion events comparing untreated cells (green dots, n = 16) against dynasore treated cells (small red dots, n = 15) is shown in the right image, evidencing synchronous fusion kinetics.

Techniques Used: Expressing, Microscopy, Fluorescence, Transfection, Transmission Assay

HIV-1 Fusion Kinetics Is Dynamin-2 Dependent in Both CD4 T Cells and TZM-bl Cells (A) Cartoon depicting the BlaM assay. Upon virion fusion and capsid release, the Vpr-BlaM chimera recognizes a FRET reporter (CCF2) that changes color (green to blue) upon cleavage. (B) Real-time BlaM was applied using HIV-1 virions packaging the Vpr-β-Lactamase chimera and pseudotyped with HXB2 Env on primary CD4 T cells at different concentrations of dynasore: 0 μM (green dots), 5 μM (black dots), 20 μM (gray dots), and 80 μM (white dots). The proportion of fusion positive cells versus total number of cells is shown (y axis) versus time, in min (x axis). (C) HIV-1/Vpr-β-Lactamase virions pseudotyped with VSVG turned out not to be fusogenic (red dots) showing the same behavior as HIV1/Vpr-β-Lactamase bald particles (without Env, black dots). (D) HIV-1 virions packaging the Vpr-β-Lactamase chimera and pseudotyped with either VSV-G (cyan dots) or JR-FL (orange dots) were exposed to TZM-bl cells with different concentrations of dynasore (0, 100, 180, 260, 340, and 400 μM) and endpoint BlaM (as defined in Experimental Procedures ) was applied. Higher concentrations of dynasore were required to fully inhibit HIV JRFL (240 μM) as compared with HIV VSVG (180 μM). (E) Time-of-addition BlaM kinetics without spinoculation protocols on HIV VSV-G virions using three different blocks: 400 μM dynasore (open red dots), temperature block (pink crosses), and NH 4 Cl (open green dots). All of the kinetics turned out to be very similar. The normalized proportion of fusion positive cells versus total number of cells is shown (y axis) versus time, in min (x axis). (F) Time-of-addition BlaM kinetics without spinoculation protocols on HIV JRFL virions using four different blocks: TAK 779 (open blue dots), dynasore (open red dots), T20 (open black dots), and temperature block (pink crosses). The normalized proportion of fusion positive cells versus total number of cells is shown (y axis) versus time, in min (x axis). In all cases, the error bars represent the SD calculated from three independent experiments.
Figure Legend Snippet: HIV-1 Fusion Kinetics Is Dynamin-2 Dependent in Both CD4 T Cells and TZM-bl Cells (A) Cartoon depicting the BlaM assay. Upon virion fusion and capsid release, the Vpr-BlaM chimera recognizes a FRET reporter (CCF2) that changes color (green to blue) upon cleavage. (B) Real-time BlaM was applied using HIV-1 virions packaging the Vpr-β-Lactamase chimera and pseudotyped with HXB2 Env on primary CD4 T cells at different concentrations of dynasore: 0 μM (green dots), 5 μM (black dots), 20 μM (gray dots), and 80 μM (white dots). The proportion of fusion positive cells versus total number of cells is shown (y axis) versus time, in min (x axis). (C) HIV-1/Vpr-β-Lactamase virions pseudotyped with VSVG turned out not to be fusogenic (red dots) showing the same behavior as HIV1/Vpr-β-Lactamase bald particles (without Env, black dots). (D) HIV-1 virions packaging the Vpr-β-Lactamase chimera and pseudotyped with either VSV-G (cyan dots) or JR-FL (orange dots) were exposed to TZM-bl cells with different concentrations of dynasore (0, 100, 180, 260, 340, and 400 μM) and endpoint BlaM (as defined in Experimental Procedures ) was applied. Higher concentrations of dynasore were required to fully inhibit HIV JRFL (240 μM) as compared with HIV VSVG (180 μM). (E) Time-of-addition BlaM kinetics without spinoculation protocols on HIV VSV-G virions using three different blocks: 400 μM dynasore (open red dots), temperature block (pink crosses), and NH 4 Cl (open green dots). All of the kinetics turned out to be very similar. The normalized proportion of fusion positive cells versus total number of cells is shown (y axis) versus time, in min (x axis). (F) Time-of-addition BlaM kinetics without spinoculation protocols on HIV JRFL virions using four different blocks: TAK 779 (open blue dots), dynasore (open red dots), T20 (open black dots), and temperature block (pink crosses). The normalized proportion of fusion positive cells versus total number of cells is shown (y axis) versus time, in min (x axis). In all cases, the error bars represent the SD calculated from three independent experiments.

Techniques Used: Blocking Assay

33) Product Images from "Antibody-induced dimerization of FGFR1 promotes receptor endocytosis independently of its kinase activity"

Article Title: Antibody-induced dimerization of FGFR1 promotes receptor endocytosis independently of its kinase activity

Journal: Scientific Reports

doi: 10.1038/s41598-017-07479-z

Internalization of FGF1 and scFvD2-Fc occurs via clathrin mediated endocytosis. Internalization of FGF1.myc ( a ) and scFvD2-Fc ( b ) into U2OSR1 cells was studied in the presence of inhibitors of dynamin (Dynasore; 80 µM) and clathrin (Pitstop2; 30 µM). Densitometric quantification of FGF1.myc and scFvD2-Fc internalization was performed with ImageJ from 3 independent experiments. Fraction of internalized FGF1.myc and scFvD2-Fc after treatment with inhibitors is represented as a percentage of untreated control. Error bars represent standard deviation. Cropped blots were displayed, full size blots are included in Supplementary Information.
Figure Legend Snippet: Internalization of FGF1 and scFvD2-Fc occurs via clathrin mediated endocytosis. Internalization of FGF1.myc ( a ) and scFvD2-Fc ( b ) into U2OSR1 cells was studied in the presence of inhibitors of dynamin (Dynasore; 80 µM) and clathrin (Pitstop2; 30 µM). Densitometric quantification of FGF1.myc and scFvD2-Fc internalization was performed with ImageJ from 3 independent experiments. Fraction of internalized FGF1.myc and scFvD2-Fc after treatment with inhibitors is represented as a percentage of untreated control. Error bars represent standard deviation. Cropped blots were displayed, full size blots are included in Supplementary Information.

Techniques Used: Standard Deviation

34) Product Images from "MyD88-dependent TLR4 signaling is selectively impaired in alveolar macrophages from asymptomatic HIV+ persons"

Article Title: MyD88-dependent TLR4 signaling is selectively impaired in alveolar macrophages from asymptomatic HIV+ persons

Journal: Blood

doi: 10.1182/blood-2009-10-250787

TLR4-mediated macrophage release of IL-10 and RANTES (but not TNFα) and IRF3 phosphorylation (but not ERK phosphorylation) depend on TLR4 endocytosis . MyD88-independent TLR4-mediated IL-10 and RANTES release requires endocytosis. (A-C) U937 and U1 macrophages were pretreated with a highly specific inhibitor of the endocytosis regulator dynamin GTPase (dynasore, 50μM) for 1 hour and then incubated in the presence or absence of lipid A (10 μg/mL) for 24 hours, and cell-free supernatants were analyzed for IL-10 (A), RANTES (B), or TNFα (C) by ELISA. Data reflect representative experiments (performed in triplicate) of 3 independent experiments with similar results. * P
Figure Legend Snippet: TLR4-mediated macrophage release of IL-10 and RANTES (but not TNFα) and IRF3 phosphorylation (but not ERK phosphorylation) depend on TLR4 endocytosis . MyD88-independent TLR4-mediated IL-10 and RANTES release requires endocytosis. (A-C) U937 and U1 macrophages were pretreated with a highly specific inhibitor of the endocytosis regulator dynamin GTPase (dynasore, 50μM) for 1 hour and then incubated in the presence or absence of lipid A (10 μg/mL) for 24 hours, and cell-free supernatants were analyzed for IL-10 (A), RANTES (B), or TNFα (C) by ELISA. Data reflect representative experiments (performed in triplicate) of 3 independent experiments with similar results. * P

Techniques Used: Incubation, Enzyme-linked Immunosorbent Assay

35) Product Images from "Giant ankyrin-G stabilizes somatodendritic GABAergic synapses through opposing endocytosis of GABAA receptors"

Article Title: Giant ankyrin-G stabilizes somatodendritic GABAergic synapses through opposing endocytosis of GABAA receptors

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

doi: 10.1073/pnas.1417989112

480-kDa ankyrin-G stabilizes extrasynaptic somatodendritic GABA A receptors through inhibition of endocytosis. ( A ) Ankyrin-G overlaps with GABA A receptor and GABARAP but is excluded from GABAergic synapses marked by gephyrin or vGAT in 3D-rendered high resolution images. (Scale bar: 1 µm in all axes.) ( B ) Dynasore (80 nM) restores GABA A receptor in ankyrin-G KO neurons. (Scale bar: 5 µm in all axes.) ** P
Figure Legend Snippet: 480-kDa ankyrin-G stabilizes extrasynaptic somatodendritic GABA A receptors through inhibition of endocytosis. ( A ) Ankyrin-G overlaps with GABA A receptor and GABARAP but is excluded from GABAergic synapses marked by gephyrin or vGAT in 3D-rendered high resolution images. (Scale bar: 1 µm in all axes.) ( B ) Dynasore (80 nM) restores GABA A receptor in ankyrin-G KO neurons. (Scale bar: 5 µm in all axes.) ** P

Techniques Used: Inhibition

36) 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

Peste des petits ruminants virus (PPRV) entry depends on low pH and dynamin. (A–C) Western blot analysis of the entry of PPRV into mock-, NH 4 Cl-, chloroquine- or dynasore-treated cells. β-actin was used as an internal control. (D) Virus titration analysis of the entry and replication of PPRV in NH 4 Cl-, chloroquine- or dynasore-treated cells. (E) CLSM analysis of TRITC-phalloidin (red), anti-PPRV (green) and DAPI (blue) in PPRV-infected EECs pre-treated with NH 4 Cl, chloroquine or dynasore. The bars indicate the mean ± SD from three independent experiments. SD, standard deviation; ∗ P
Figure Legend Snippet: Peste des petits ruminants virus (PPRV) entry depends on low pH and dynamin. (A–C) Western blot analysis of the entry of PPRV into mock-, NH 4 Cl-, chloroquine- or dynasore-treated cells. β-actin was used as an internal control. (D) Virus titration analysis of the entry and replication of PPRV in NH 4 Cl-, chloroquine- or dynasore-treated cells. (E) CLSM analysis of TRITC-phalloidin (red), anti-PPRV (green) and DAPI (blue) in PPRV-infected EECs pre-treated with NH 4 Cl, chloroquine or dynasore. The bars indicate the mean ± SD from three independent experiments. SD, standard deviation; ∗ P

Techniques Used: Western Blot, Titration, Confocal Laser Scanning Microscopy, Infection, Standard Deviation

37) Product Images from "Essential Role for Endocytosis in the Growth Factor-stimulated Activation of ERK1/2 in Endothelial Cells *"

Article Title: Essential Role for Endocytosis in the Growth Factor-stimulated Activation of ERK1/2 in Endothelial Cells *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M112.446401

Small molecule inhibitors of internalization suppress angiogenesis. A–C , beads coated with adherent HUVECs were embedded in a fibrinogen gel and then cultured in the presence of growth factors (VEGF and FGF2) and vehicle or growth factors and the indicated concentrations of drug for 7 days. Graphs in A and B show the number of tubules formed per bead quantified after 7 days ± S.E. Quantification of 20 beads from 2 independent experiments was used to generate each data point. Examples of beads treated as indicated after 7 days are shown in C . Beads are stained with rhodamine-phalloidin ( red ) and DAPI ( blue ). D and E , sponges were subcutaneously implanted into mice and were injected three times per week with vehicle alone, dynasore alone, growth factors and vehicle (VEGF, FGF2, and vehicle), or growth factors and dynasore (VEGF, FGF2, and dynasore). After 21 days sponges were removed for analysis of blood vessel infiltration by staining for endomucin. The graph in D shows the number of blood vessels per mm 2 quantified in sponges from mice treated as indicated ( n = 6 mice per treatment group). Representative images of the endomucin staining are shown in E. *, p
Figure Legend Snippet: Small molecule inhibitors of internalization suppress angiogenesis. A–C , beads coated with adherent HUVECs were embedded in a fibrinogen gel and then cultured in the presence of growth factors (VEGF and FGF2) and vehicle or growth factors and the indicated concentrations of drug for 7 days. Graphs in A and B show the number of tubules formed per bead quantified after 7 days ± S.E. Quantification of 20 beads from 2 independent experiments was used to generate each data point. Examples of beads treated as indicated after 7 days are shown in C . Beads are stained with rhodamine-phalloidin ( red ) and DAPI ( blue ). D and E , sponges were subcutaneously implanted into mice and were injected three times per week with vehicle alone, dynasore alone, growth factors and vehicle (VEGF, FGF2, and vehicle), or growth factors and dynasore (VEGF, FGF2, and dynasore). After 21 days sponges were removed for analysis of blood vessel infiltration by staining for endomucin. The graph in D shows the number of blood vessels per mm 2 quantified in sponges from mice treated as indicated ( n = 6 mice per treatment group). Representative images of the endomucin staining are shown in E. *, p

Techniques Used: Cell Culture, Staining, Mouse Assay, Injection

38) Product Images from "Japanese Encephalitis Virus Infects Neuronal Cells through a Clathrin-Independent Endocytic Mechanism"

Article Title: Japanese Encephalitis Virus Infects Neuronal Cells through a Clathrin-Independent Endocytic Mechanism

Journal: Journal of Virology

doi: 10.1128/JVI.01399-12

JEV internalization is dynamin dependent. (A) Neuro2a cells grown on coverslips were either untreated or treated with 80 μM dynasore for 1 h, following which they were infected with JEV (MOI, 0.4) in the presence of the inhibitor. At 24 hpi cells
Figure Legend Snippet: JEV internalization is dynamin dependent. (A) Neuro2a cells grown on coverslips were either untreated or treated with 80 μM dynasore for 1 h, following which they were infected with JEV (MOI, 0.4) in the presence of the inhibitor. At 24 hpi cells

Techniques Used: Infection

39) Product Images from "Glycosylated extracellular vesicles released by glioblastoma cells are decorated by CCL18 allowing for cellular uptake via chemokine receptor CCR8"

Article Title: Glycosylated extracellular vesicles released by glioblastoma cells are decorated by CCL18 allowing for cellular uptake via chemokine receptor CCR8

Journal: Journal of Extracellular Vesicles

doi: 10.1080/20013078.2018.1446660

CCR8 acts as an EV receptor (a) GPCR siRNA screening result on HEK-293T cells. Normalised PKH67 fluorescence is displayed on the Y-axis, and each dot represents a siRNA against an individual GPCR Top-8 EV uptake-reducing siRNAs are highlighted in orange and indicated at the right side. (b) siRNA screening validation on GBM8 cells with an independent set of siRNAs. Normalised PKH67 fluorescence (EV uptake) is displayed on the Y-axis. Treatment of cells with Dynasore served as a control for EV uptake reduction. (c) CCR8 inhibitor MC148 reduces EV uptake in a dose-dependent fashion. (d) FACS analysis of CCR8 expression in GBM8 cells. Negative control: No anti-CCR8 primary antibody (e) PKH67 fluorescence (representative of EV uptake) of CCR8 − (green) compared to CCR8 + (orange) GBM8 cells. Negative control (grey): GBM8 cells not incubated with PKH67 EVs.
Figure Legend Snippet: CCR8 acts as an EV receptor (a) GPCR siRNA screening result on HEK-293T cells. Normalised PKH67 fluorescence is displayed on the Y-axis, and each dot represents a siRNA against an individual GPCR Top-8 EV uptake-reducing siRNAs are highlighted in orange and indicated at the right side. (b) siRNA screening validation on GBM8 cells with an independent set of siRNAs. Normalised PKH67 fluorescence (EV uptake) is displayed on the Y-axis. Treatment of cells with Dynasore served as a control for EV uptake reduction. (c) CCR8 inhibitor MC148 reduces EV uptake in a dose-dependent fashion. (d) FACS analysis of CCR8 expression in GBM8 cells. Negative control: No anti-CCR8 primary antibody (e) PKH67 fluorescence (representative of EV uptake) of CCR8 − (green) compared to CCR8 + (orange) GBM8 cells. Negative control (grey): GBM8 cells not incubated with PKH67 EVs.

Techniques Used: Fluorescence, FACS, Expressing, Negative Control, Incubation

40) Product Images from "Persistent cAMP-Signals Triggered by Internalized G-Protein-Coupled Receptors"

Article Title: Persistent cAMP-Signals Triggered by Internalized G-Protein-Coupled Receptors

Journal: PLoS Biology

doi: 10.1371/journal.pbio.1000172

Effect of endocytosis inhibition on downstream signaling. (A and B) Actin depolymerization in response to TSH. Mouse primary thyroid cells were preincubated with normal medium or medium plus 80 µM dynasore for 20 min and stimulated with 30 U/l TSH for an additional 20 min in the presence or absence of dynasore as indicated. Cells were then fixed, and actin was stained with fluorescent phalloidin. Note that dynasore largely prevented the depolymerization of actin in response to TSH. (B) High-magnification images of actin rearrangement in lamellipodia, where the effect of dynasore was more pronounced. (C) VASP phosphorylation. Primary mouse thyroid cells were preincubated with normal medium or medium plus 80 µM dynasore for 20 min. Cells were then stimulated with 1 U/l TSH for 30 min, in the presence or absence of dynasore as indicated. Levels of P-VASP (Ser 157) and total VASP were evaluated by Western blot analysis. Shown are the mean P-VASP levels of three independent experiments. Error bars indicate SEM. (D) Subcellular localization of VASP. Mouse primary thyroid cells were labeled by immunofluorescence with an antibody against total VASP (red) together with fluorescent phalloidin to stain actin (green). Shown is a merged fluorescent image. VASP is typically located at the ends of actin filaments. (E) Pattern of VASP phosphorylation in response to TSH. Mouse primary thyroid cells were preincubated and stimulated with TSH in the presence or absence of dynasore as explained above. Cells were then labeled by immunofluorescence with an antibody against VASP phosphorylated at Ser 157 (red) together with fluorescent phalloidin to stain actin (green). Note the appearance of spots containing phosphorylated VASP and actin in the central cellular compartment only in the absence of dynasore. (F) Actin depolymerization and pattern of VASP phosphorylation in response to forskolin. Cells were treated as in (E), with the exception that instead of TSH, they were stimulated with 10 µM forskolin. Note a similar degree of actin depolymerization and a similar pattern of VASP phosphorylation both in the presence and in the absence of dynasore. Images in (A and B) and (D–F) are representative of at least three independent experiments.
Figure Legend Snippet: Effect of endocytosis inhibition on downstream signaling. (A and B) Actin depolymerization in response to TSH. Mouse primary thyroid cells were preincubated with normal medium or medium plus 80 µM dynasore for 20 min and stimulated with 30 U/l TSH for an additional 20 min in the presence or absence of dynasore as indicated. Cells were then fixed, and actin was stained with fluorescent phalloidin. Note that dynasore largely prevented the depolymerization of actin in response to TSH. (B) High-magnification images of actin rearrangement in lamellipodia, where the effect of dynasore was more pronounced. (C) VASP phosphorylation. Primary mouse thyroid cells were preincubated with normal medium or medium plus 80 µM dynasore for 20 min. Cells were then stimulated with 1 U/l TSH for 30 min, in the presence or absence of dynasore as indicated. Levels of P-VASP (Ser 157) and total VASP were evaluated by Western blot analysis. Shown are the mean P-VASP levels of three independent experiments. Error bars indicate SEM. (D) Subcellular localization of VASP. Mouse primary thyroid cells were labeled by immunofluorescence with an antibody against total VASP (red) together with fluorescent phalloidin to stain actin (green). Shown is a merged fluorescent image. VASP is typically located at the ends of actin filaments. (E) Pattern of VASP phosphorylation in response to TSH. Mouse primary thyroid cells were preincubated and stimulated with TSH in the presence or absence of dynasore as explained above. Cells were then labeled by immunofluorescence with an antibody against VASP phosphorylated at Ser 157 (red) together with fluorescent phalloidin to stain actin (green). Note the appearance of spots containing phosphorylated VASP and actin in the central cellular compartment only in the absence of dynasore. (F) Actin depolymerization and pattern of VASP phosphorylation in response to forskolin. Cells were treated as in (E), with the exception that instead of TSH, they were stimulated with 10 µM forskolin. Note a similar degree of actin depolymerization and a similar pattern of VASP phosphorylation both in the presence and in the absence of dynasore. Images in (A and B) and (D–F) are representative of at least three independent experiments.

Techniques Used: Inhibition, Staining, Western Blot, Labeling, Immunofluorescence

Effect of endocytosis inhibition on cAMP signaling. Cells were prestimulated with 0.43 M sucrose for 10 min, 80 µM dynasore for 20 min, or normal medium as control. (A) Comparison of FRET changes induced by stimulating thyroid follicles obtained from CAG-Epac1-camps mice with TSH (30 U/l for 2 min, as in Figure 5C ) in the presence or absence (control) of endocytosis inhibitors (n = 6–8 per each condition). Error bars indicate SEM. (B) Confocal image of a primary mouse thyroid cell stimulated with TSH-Alexa594 (3 µg/ml for 20 min) in the presence of 0.43 M sucrose. Note the binding of TSH-Alexa594 to the plasma membrane (arrowheads) and the almost complete inhibition of TSH-Alexa594 internalization (no intracellular vesicles). For comparison, see Figure 8 (20 min). (C) Comparison of cAMP signal reversibility after TSH stimulation (30 U/l for 2 min) in the presence or absence (control) of 0.43 M sucrose (n = 6, each). (D) Confocal image of a primary mouse thyroid cell stimulated with TSH-Alexa594 (3 µg/ml for 20 min) in the presence of 80 µM dynasore, showing consistent inhibition of TSH-Alexa594 internalization. Arrowheads, TSH-Alexa594 bound to the plasma membrane. (E) Comparison of cAMP signal reversibility after TSH stimulation (30 U/l for 2 min) in the presence or absence (control) of 80 µM dynasore (n = 6, control; n = 8, dynasore). Signal reversibility in (C) and (E) is calculated as in Figure 5F . Fits were compared with F test, having a null hypothesis that Y max values were the same for all datasets. Images in (B) and (D) are representative of more than 20 cells per condition analyzed in three independent experiments.
Figure Legend Snippet: Effect of endocytosis inhibition on cAMP signaling. Cells were prestimulated with 0.43 M sucrose for 10 min, 80 µM dynasore for 20 min, or normal medium as control. (A) Comparison of FRET changes induced by stimulating thyroid follicles obtained from CAG-Epac1-camps mice with TSH (30 U/l for 2 min, as in Figure 5C ) in the presence or absence (control) of endocytosis inhibitors (n = 6–8 per each condition). Error bars indicate SEM. (B) Confocal image of a primary mouse thyroid cell stimulated with TSH-Alexa594 (3 µg/ml for 20 min) in the presence of 0.43 M sucrose. Note the binding of TSH-Alexa594 to the plasma membrane (arrowheads) and the almost complete inhibition of TSH-Alexa594 internalization (no intracellular vesicles). For comparison, see Figure 8 (20 min). (C) Comparison of cAMP signal reversibility after TSH stimulation (30 U/l for 2 min) in the presence or absence (control) of 0.43 M sucrose (n = 6, each). (D) Confocal image of a primary mouse thyroid cell stimulated with TSH-Alexa594 (3 µg/ml for 20 min) in the presence of 80 µM dynasore, showing consistent inhibition of TSH-Alexa594 internalization. Arrowheads, TSH-Alexa594 bound to the plasma membrane. (E) Comparison of cAMP signal reversibility after TSH stimulation (30 U/l for 2 min) in the presence or absence (control) of 80 µM dynasore (n = 6, control; n = 8, dynasore). Signal reversibility in (C) and (E) is calculated as in Figure 5F . Fits were compared with F test, having a null hypothesis that Y max values were the same for all datasets. Images in (B) and (D) are representative of more than 20 cells per condition analyzed in three independent experiments.

Techniques Used: Inhibition, Mouse Assay, Binding Assay

Related Articles

Electron Microscopy:

Article Title: Brain-specific Drp1 regulates postsynaptic endocytosis and dendrite formation independently of mitochondrial division
Article Snippet: .. For dynasore treatment, cells were incubated with 80 µM of dynasore (Sigma-Aldrich, D7693) in culture medium for different times, then fixed and further processed for electron microscopy as described above. .. To stimulate endocytosis through chemical long-term depression (chemical LTD), neurons were incubated with 20 µM of NMDA (Tocris, 0114), 10 µM of glycine (Tocris, 0219), 0.3 mM of MgCl2 , 2 mM of CaCl2 and 1 µM of TTX (Tocris, 1078) in Base buffer (10 mM HEPES, pH 7.4, 140 mM NaCl, 2.4 mM KCl, 10 mM glucose) for 4 min. As a control, Base buffer containing 2 mM of MgCl2 , 2 mM of CaCl2 and 1 µM of TTX was used.

In Vitro:

Article Title: Impact of Dynasore an Inhibitor of Dynamin II on Shigella flexneri Infection
Article Snippet: .. Chemicals and antibodies Dynasore (EC-000.2052; Exclusive Chemistry, D7693-25MG; Sigma-Aldrich, ab120192; Abcam) was prepared as an 80 mM stock in dimethyl sulfoxide (DMSO) (D2650; Sigma-Aldrich) for in vitro studies. .. For in vivo studies, dynasore was dissolved in a formulation containing 1-methyl-2-pyrrolidione (NMP/Pharmasolve; Ashland ISP) and polyethylene glycol 300 (PEG300; Sigma-Aldrich) (1 part NMP to 9 parts PEG300).

Blocking Assay:

Article Title: Retinal Pigment Epithelial Cells Mitigate the Effects of Complement Attack by Endocytosis of C5b-9
Article Snippet: .. To block endocytosis of the C5b-9 complex, media was supplemented with 200 μg/ml Dynasore hydrate (Sigma; D7693) for 24 h (unless stated otherwise) at 37°C/5% CO2 . .. The next day, cells were washed once with sterile PBS and fixed for immunofluorescence or electron microscopy.

Concentration Assay:

Article Title: Cisplatin induces the release of extracellular vesicles from ovarian cancer cells that can induce invasiveness and drug resistance in bystander cells
Article Snippet: .. Dynasore (Sigma, D7693) was diluted in DMSO to a concentration of 31 mM, stored at −20°C and added to cells at a concentration of 50 µM; 30 min after the drug treatment, cisplatin was added at varying concentrations to give a cisplatin response curve. ..

Article Title: Novel mechanism regulating endothelial permeability via T-cadherin-dependent VE-cadherin phosphorylation and clathrin-mediated endocytosis
Article Snippet: .. In internalization assay HUVEC were preincubated with dynasore hydrate (Sigma) in final concentration 80 μM for 2 h, or LysoTracker® Red (Invitrogen, Ex/Em: 577/590 nm) for 60 and 120 min, or Y27632 in final concentration 10 μM (Sigma) for 12 h before immunofluorescent staining. .. Images were acquired by confocal laser scanning microscopy using a microscope system (TCS SP5, Leica) equipped with a Plan-Apo ×60, 1.40 NA oil objective and 488- and 543- Argon lasers lines.

Incubation:

Article Title: Brain-specific Drp1 regulates postsynaptic endocytosis and dendrite formation independently of mitochondrial division
Article Snippet: .. For dynasore treatment, cells were incubated with 80 µM of dynasore (Sigma-Aldrich, D7693) in culture medium for different times, then fixed and further processed for electron microscopy as described above. .. To stimulate endocytosis through chemical long-term depression (chemical LTD), neurons were incubated with 20 µM of NMDA (Tocris, 0114), 10 µM of glycine (Tocris, 0219), 0.3 mM of MgCl2 , 2 mM of CaCl2 and 1 µM of TTX (Tocris, 1078) in Base buffer (10 mM HEPES, pH 7.4, 140 mM NaCl, 2.4 mM KCl, 10 mM glucose) for 4 min. As a control, Base buffer containing 2 mM of MgCl2 , 2 mM of CaCl2 and 1 µM of TTX was used.

Inhibition:

Article Title: Dynamin2 controls Rap1 activation and integrin clustering in human T lymphocyte adhesion
Article Snippet: .. Chemical inhibitors The following small molecules were used for the chemical inhibition of specific proteins or cellular processes: brefeldin A (B6542, Sigma-Aldrich), chlorpromazine (C8138, Sigma-Aldrich), cytochalasin D (C8273, Sigma-Aldrich), dynasore hydrate (D7693, Sigma-Aldrich), dynole 34–2 (ab120463, Abcam), dynole 31–2 (ab120464, Abcam), Exo1 (#1850, Tocris), latrunculin A (L5163, Sigma-Aldrich), monodansylcadaverine (30432, Sigma-Aldrich). .. Except for Brefeldin A and Chlorpromazine, which were diluted in ethanol or water respectively, all stock solutions were prepared using dimethyl sulfoxide (DMSO, #276855, Sigma-Aldrich).

Article Title: Inhibitors of endocytosis prevent Wnt/Wingless signalling by reducing the level of basal β-catenin/Armadillo
Article Snippet: .. Endocytosis inhibition S2R+ or RKO cells were treated with Dynasore (200 µM, Sigma-Aldrich D7693), Dyngo-4a (100 µM, Abcam ab120689), or DMSO as a control, for 30 minutes before stimulation with Wingless- or Wnt-3A-conditioned medium for the indicated times. ..

Staining:

Article Title: Novel mechanism regulating endothelial permeability via T-cadherin-dependent VE-cadherin phosphorylation and clathrin-mediated endocytosis
Article Snippet: .. In internalization assay HUVEC were preincubated with dynasore hydrate (Sigma) in final concentration 80 μM for 2 h, or LysoTracker® Red (Invitrogen, Ex/Em: 577/590 nm) for 60 and 120 min, or Y27632 in final concentration 10 μM (Sigma) for 12 h before immunofluorescent staining. .. Images were acquired by confocal laser scanning microscopy using a microscope system (TCS SP5, Leica) equipped with a Plan-Apo ×60, 1.40 NA oil objective and 488- and 543- Argon lasers lines.

other:

Article Title: The cytotoxicity of the α1-adrenoceptor antagonist prazosin is linked to an endocytotic mechanism equivalent to transport-P
Article Snippet: Drugs Bafilomycin A1, chloroquine diphosphate salt, dynasore hydrate, methyl-β-cyclodextrin and prazosin hydrochloride were purchased from Sigma–Aldrich.

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 85
    Millipore dynamin inhibitory compounds dynasore
    Lamellipodia formation and dynamics in pancreatic tumor cells are dependent upon Dyn2 expression and phosphorylation (a) Western blot analysis of stable BxPC-3 cell lines expressing either GFP alone, Dyn2-GFP, or Dyn2Y(231,597)F-GFP. (b-d) Control BxPC-3 cells stained for cortactin (b) grow in tight clusters and appear non-motile, while stable lines expressing Dyn2-GFP (c) exhibit numerous dynamic lamellipodia. (d) In comparison, mutant Dyn2Y(231,597)F-GFP expressing cells appear loosely clustered with some motile morphology. (c-d) show GFP fluorescence of the Dyn2-GFP and Dyn2Y(231,597)F-GFP. (e) Kymographs generated from movies of the lamellipodia of BxPC-3 stable cells showing the rapid protrusion rate of lamellipodia in cells expressing WT Dyn2-GFP, relative to cells expressing GFP or Dyn2Y(231/597)F -GFP. (f) Average lamellipodia protrusion speed calculated from more than 11 cells per cell line based on collected kymographs shown in (e). (g) Western blot on lysates from BxPC-3-GFP cells treated with non-targeting siRNA (NTsi) or Dyn2 siRNA (D2si). Kymographs (i) and quantitation (i) showing reduced lamellipodia extension following Dyn2 knockdown. Kymographs (j) and quantitation (k) showing lamellipodia extension of BxPC-3-GFP cells following treatment with DMSO or <t>dynamin</t> inhibitors <t>Dynasore</t> (80 μM) or MiTMAB (20 μM). Error bars represent S.E.M. Student’s t-test was used for statistics (** represents p
    Dynamin Inhibitory Compounds Dynasore, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dynamin inhibitory compounds dynasore/product/Millipore
    Average 85 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    dynamin inhibitory compounds dynasore - by Bioz Stars, 2020-09
    85/100 stars
      Buy from Supplier

    98
    Millipore dynasore
    Pharmacological and genetic inhibition of Dyn2 function also reduces starvation-induced LD breakdown. HuH-7 (A) and Hep3B (B) hepatocytes were loaded with 150 µM oleate overnight and starved for 48 h in medium containing 0.1% FBS in the presence of Dyn2 inhibitors or DMSO as indicated. Representative images of inhibitor-treated and control cells (stained with Oil Red O) are shown in A and B together with the quantitation of the average LD area per cell from three independent experiments. Pharmacological inhibitors used were: <t>Dynasore</t> (inhibits Dyn2 GTPase activity), MiTMAB (targets PH domain and interferes with membrane binding), Dynole 34-2 (allosteric GTPase inhibitor), and Dynole 31-2 (negative control for Dynole 34-2). Bars, 20 µM. (C) Representative images from control and Dyn2 knockout MEFs after an overnight lipid loading with 400 µM oleate for 17 h. Knockout of Dyn2 was induced by treatment with 2 µM 4-hydroxy-tamoxifen for 7 d and was confirmed by immunostaining of endogenous Dyn2 (top row) and by immunoblot (D). Bars, 20 µM. (E and F) Average LD number (E) and area (F) per cell from five independent experiments. All data are represented as mean ± SE. *, P ≤ 0.05; **, P ≤ 0.01. (G) Whole-cell lysates and LD fractions isolated from HuH-7 hepatocytes under resting or starved (2 h HBSS starvation) conditions. (H) Primary hepatocyte expressing Dyn2-GFP, showing an absence of colocalization with the LD surface (stained with Oil Red O). Bar, 20 µM. Inset shows magnification of boxed region (bar, 2 µM).
    Dynasore, supplied by Millipore, used in various techniques. Bioz Stars score: 98/100, based on 153 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dynasore/product/Millipore
    Average 98 stars, based on 153 article reviews
    Price from $9.99 to $1999.99
    dynasore - by Bioz Stars, 2020-09
    98/100 stars
      Buy from Supplier

    Image Search Results


    Lamellipodia formation and dynamics in pancreatic tumor cells are dependent upon Dyn2 expression and phosphorylation (a) Western blot analysis of stable BxPC-3 cell lines expressing either GFP alone, Dyn2-GFP, or Dyn2Y(231,597)F-GFP. (b-d) Control BxPC-3 cells stained for cortactin (b) grow in tight clusters and appear non-motile, while stable lines expressing Dyn2-GFP (c) exhibit numerous dynamic lamellipodia. (d) In comparison, mutant Dyn2Y(231,597)F-GFP expressing cells appear loosely clustered with some motile morphology. (c-d) show GFP fluorescence of the Dyn2-GFP and Dyn2Y(231,597)F-GFP. (e) Kymographs generated from movies of the lamellipodia of BxPC-3 stable cells showing the rapid protrusion rate of lamellipodia in cells expressing WT Dyn2-GFP, relative to cells expressing GFP or Dyn2Y(231/597)F -GFP. (f) Average lamellipodia protrusion speed calculated from more than 11 cells per cell line based on collected kymographs shown in (e). (g) Western blot on lysates from BxPC-3-GFP cells treated with non-targeting siRNA (NTsi) or Dyn2 siRNA (D2si). Kymographs (i) and quantitation (i) showing reduced lamellipodia extension following Dyn2 knockdown. Kymographs (j) and quantitation (k) showing lamellipodia extension of BxPC-3-GFP cells following treatment with DMSO or dynamin inhibitors Dynasore (80 μM) or MiTMAB (20 μM). Error bars represent S.E.M. Student’s t-test was used for statistics (** represents p

    Journal: Oncogene

    Article Title: Increased Expression of the Large GTPase Dynamin 2 Potentiates Metastatic Migration and Invasion of Pancreatic Ductal Carcinoma

    doi: 10.1038/onc.2011.329

    Figure Lengend Snippet: Lamellipodia formation and dynamics in pancreatic tumor cells are dependent upon Dyn2 expression and phosphorylation (a) Western blot analysis of stable BxPC-3 cell lines expressing either GFP alone, Dyn2-GFP, or Dyn2Y(231,597)F-GFP. (b-d) Control BxPC-3 cells stained for cortactin (b) grow in tight clusters and appear non-motile, while stable lines expressing Dyn2-GFP (c) exhibit numerous dynamic lamellipodia. (d) In comparison, mutant Dyn2Y(231,597)F-GFP expressing cells appear loosely clustered with some motile morphology. (c-d) show GFP fluorescence of the Dyn2-GFP and Dyn2Y(231,597)F-GFP. (e) Kymographs generated from movies of the lamellipodia of BxPC-3 stable cells showing the rapid protrusion rate of lamellipodia in cells expressing WT Dyn2-GFP, relative to cells expressing GFP or Dyn2Y(231/597)F -GFP. (f) Average lamellipodia protrusion speed calculated from more than 11 cells per cell line based on collected kymographs shown in (e). (g) Western blot on lysates from BxPC-3-GFP cells treated with non-targeting siRNA (NTsi) or Dyn2 siRNA (D2si). Kymographs (i) and quantitation (i) showing reduced lamellipodia extension following Dyn2 knockdown. Kymographs (j) and quantitation (k) showing lamellipodia extension of BxPC-3-GFP cells following treatment with DMSO or dynamin inhibitors Dynasore (80 μM) or MiTMAB (20 μM). Error bars represent S.E.M. Student’s t-test was used for statistics (** represents p

    Article Snippet: Dynamin inhibitory compounds Dynasore and MiTMAB, from Calbiochem (Cat # 324410 and 324411), were resuspended in dimethyl sulfoxide (DMSO) to 25 mM stock concentrations and stored at −20°C until use.

    Techniques: Expressing, Western Blot, Staining, Mutagenesis, Fluorescence, Generated, Quantitation Assay

    Dynamin function is required for cell migration during wound healing Confluent monolayers of the motile pancreatic cancer cell line Panc04.03 were wounded and stimulated with 50 ng/mL EGF in the presence or absence of the indicated doses of the dynamin inhibitory compounds MiTMAB (a, b) or Dynasore (c, d), or after treatment with non-targeting (NT) or Dyn2 (D2) siRNA (e, f). Phase images of cell monolayers treated with DMSO (a,c), 20 μM MiTMAB (a), or 80 μM Dynasore (c) at 1 and 5 hrs post-EGF stimulation. Increasing the dosage of either drug resulted in a dose-dependent reduction in cell migration, contributing to slower wound closure. Similarly, depletion of Dyn2 by siRNA significantly reduced cell migration speed (e, f). Efficient knockdown of Dyn2 is shown in the inset in panel (f). The average speed of the wound edge from 3 experiments are shown. Error bars represent S.E.M. Student’s t-test was used for statistics (** represents p

    Journal: Oncogene

    Article Title: Increased Expression of the Large GTPase Dynamin 2 Potentiates Metastatic Migration and Invasion of Pancreatic Ductal Carcinoma

    doi: 10.1038/onc.2011.329

    Figure Lengend Snippet: Dynamin function is required for cell migration during wound healing Confluent monolayers of the motile pancreatic cancer cell line Panc04.03 were wounded and stimulated with 50 ng/mL EGF in the presence or absence of the indicated doses of the dynamin inhibitory compounds MiTMAB (a, b) or Dynasore (c, d), or after treatment with non-targeting (NT) or Dyn2 (D2) siRNA (e, f). Phase images of cell monolayers treated with DMSO (a,c), 20 μM MiTMAB (a), or 80 μM Dynasore (c) at 1 and 5 hrs post-EGF stimulation. Increasing the dosage of either drug resulted in a dose-dependent reduction in cell migration, contributing to slower wound closure. Similarly, depletion of Dyn2 by siRNA significantly reduced cell migration speed (e, f). Efficient knockdown of Dyn2 is shown in the inset in panel (f). The average speed of the wound edge from 3 experiments are shown. Error bars represent S.E.M. Student’s t-test was used for statistics (** represents p

    Article Snippet: Dynamin inhibitory compounds Dynasore and MiTMAB, from Calbiochem (Cat # 324410 and 324411), were resuspended in dimethyl sulfoxide (DMSO) to 25 mM stock concentrations and stored at −20°C until use.

    Techniques: Migration

    Elevated dynamin expression significantly increases individual tumor cell migration (a) Low magnification phase images of the BxPC-3 stable cell lines described in Fig 2 were acquired over 11 hrs and subsequently traced to produce migration paths. The black line depicts the migratory path of a cell over the observation period. (b) Migration tracks generated from stably transfected cells expressing GFP, (c) Dyn2-GFP and (d) Dyn2Y(231/597)F -GFP. (e) Graph depicting total migration distance of tumor cells stably overexpressing GFP, Dyn2-GFP, or Dyn2Y(231/597)F -GFP (e), Dyn2-GFP-expressing cells treated with the dynamin inhibitors MiTMAB or Dynasore at the indicated concentrations (f), or control GFP-expressing cells treated with dynamin inhibitors (g) or siRNA directed against Dyn2 (h). Compared to the other cell lines, the tumor cell line expressing Dyn2-GFP exhibited a significant increase in migration that was markedly reduced by inhibiting dynamin function. Further inhibition of endogenous Dyn2 in control cells using inhibitory compounds or Dyn2 depletion also reduced cell migration. (n=100 cells per condition, average of three experiments). Student’s t-test was used for statistics (*** represents p

    Journal: Oncogene

    Article Title: Increased Expression of the Large GTPase Dynamin 2 Potentiates Metastatic Migration and Invasion of Pancreatic Ductal Carcinoma

    doi: 10.1038/onc.2011.329

    Figure Lengend Snippet: Elevated dynamin expression significantly increases individual tumor cell migration (a) Low magnification phase images of the BxPC-3 stable cell lines described in Fig 2 were acquired over 11 hrs and subsequently traced to produce migration paths. The black line depicts the migratory path of a cell over the observation period. (b) Migration tracks generated from stably transfected cells expressing GFP, (c) Dyn2-GFP and (d) Dyn2Y(231/597)F -GFP. (e) Graph depicting total migration distance of tumor cells stably overexpressing GFP, Dyn2-GFP, or Dyn2Y(231/597)F -GFP (e), Dyn2-GFP-expressing cells treated with the dynamin inhibitors MiTMAB or Dynasore at the indicated concentrations (f), or control GFP-expressing cells treated with dynamin inhibitors (g) or siRNA directed against Dyn2 (h). Compared to the other cell lines, the tumor cell line expressing Dyn2-GFP exhibited a significant increase in migration that was markedly reduced by inhibiting dynamin function. Further inhibition of endogenous Dyn2 in control cells using inhibitory compounds or Dyn2 depletion also reduced cell migration. (n=100 cells per condition, average of three experiments). Student’s t-test was used for statistics (*** represents p

    Article Snippet: Dynamin inhibitory compounds Dynasore and MiTMAB, from Calbiochem (Cat # 324410 and 324411), were resuspended in dimethyl sulfoxide (DMSO) to 25 mM stock concentrations and stored at −20°C until use.

    Techniques: Expressing, Migration, Stable Transfection, Generated, Transfection, Inhibition

    Dyn2 function is essential for chemotactic invasion in vitro Fluorescence images of tumor cells plated on transwell filters. 2×10 5 stable BxPC-3 cells expressing GFP (a), Dyn2-GFP (b) or Dyn2Y(231/597)F -GFP (c), were plated in low serum medium (0.2%) in the top of a blind-well chamber, separated from high serum (10% FBS) in the bottom chamber by a gelatin-coated filter containing 8 μm pores and returned to the culture incubator for 4 hrs. After fixation, the cells on the filters were co-stained with phalloidin for actin (white) to visualize cell bodies and DAPI (blue) to visualize nuclei. The image focal plane in a-f represents the bottom of each filter and reveals cells that have successfully translocated by the emergence of a blue nucleus. While numerous Dyn2-GFP expressing cells have migrated through the filter (b), most cells expressing the GFP or Dyn2Y(231/597)F-GFP protein remain at the top of the chamber (a,c). These same confocal images were reoriented as brightest point projection Z-series, with the filter indicated by the yellow double lines (a’-c’). Note the marked increase in nuclei of Dyn2-GFP expressing cells below the filter compared to cells expressing Dyn2Y(231,597)F-GFP or the control. (g) Quantification of the percentage of cells that crossed a gelatin-coated filter in blind-well assays. Notably, twice as many Dyn2-GFP-expressing cells migrated across the filter as did cells expressing GFP, and more than 3 times the number compared to Dyn2Y(231/597)F-GFP. (d-f) Confocal images of the same Dyn2-GFP expressing cell line plated on filters as above and treated with DMSO as a control or the dynamin inhibitory compounds Dynasore (50 μM) and MiTMAB (10 μM). Migratory invasion through the filters was almost completely prevented in the presence of dynamin inhibitory compounds MiTMAB and Dynasore as shown clearly in the Z-series confocal images of these fields (d’-f’) and quantitated in (h). Similarly, the basal level of migration in GFP-expressing control cells was attenuated after depletion (i) or small-molecule inhibition of (j) endogenous Dyn2. Average +/− S.E.M of greater than 150 cells per line. Student’s t-test was used for statistics (**represents p

    Journal: Oncogene

    Article Title: Increased Expression of the Large GTPase Dynamin 2 Potentiates Metastatic Migration and Invasion of Pancreatic Ductal Carcinoma

    doi: 10.1038/onc.2011.329

    Figure Lengend Snippet: Dyn2 function is essential for chemotactic invasion in vitro Fluorescence images of tumor cells plated on transwell filters. 2×10 5 stable BxPC-3 cells expressing GFP (a), Dyn2-GFP (b) or Dyn2Y(231/597)F -GFP (c), were plated in low serum medium (0.2%) in the top of a blind-well chamber, separated from high serum (10% FBS) in the bottom chamber by a gelatin-coated filter containing 8 μm pores and returned to the culture incubator for 4 hrs. After fixation, the cells on the filters were co-stained with phalloidin for actin (white) to visualize cell bodies and DAPI (blue) to visualize nuclei. The image focal plane in a-f represents the bottom of each filter and reveals cells that have successfully translocated by the emergence of a blue nucleus. While numerous Dyn2-GFP expressing cells have migrated through the filter (b), most cells expressing the GFP or Dyn2Y(231/597)F-GFP protein remain at the top of the chamber (a,c). These same confocal images were reoriented as brightest point projection Z-series, with the filter indicated by the yellow double lines (a’-c’). Note the marked increase in nuclei of Dyn2-GFP expressing cells below the filter compared to cells expressing Dyn2Y(231,597)F-GFP or the control. (g) Quantification of the percentage of cells that crossed a gelatin-coated filter in blind-well assays. Notably, twice as many Dyn2-GFP-expressing cells migrated across the filter as did cells expressing GFP, and more than 3 times the number compared to Dyn2Y(231/597)F-GFP. (d-f) Confocal images of the same Dyn2-GFP expressing cell line plated on filters as above and treated with DMSO as a control or the dynamin inhibitory compounds Dynasore (50 μM) and MiTMAB (10 μM). Migratory invasion through the filters was almost completely prevented in the presence of dynamin inhibitory compounds MiTMAB and Dynasore as shown clearly in the Z-series confocal images of these fields (d’-f’) and quantitated in (h). Similarly, the basal level of migration in GFP-expressing control cells was attenuated after depletion (i) or small-molecule inhibition of (j) endogenous Dyn2. Average +/− S.E.M of greater than 150 cells per line. Student’s t-test was used for statistics (**represents p

    Article Snippet: Dynamin inhibitory compounds Dynasore and MiTMAB, from Calbiochem (Cat # 324410 and 324411), were resuspended in dimethyl sulfoxide (DMSO) to 25 mM stock concentrations and stored at −20°C until use.

    Techniques: In Vitro, Fluorescence, Expressing, Staining, Migration, Inhibition

    Pharmacological and genetic inhibition of Dyn2 function also reduces starvation-induced LD breakdown. HuH-7 (A) and Hep3B (B) hepatocytes were loaded with 150 µM oleate overnight and starved for 48 h in medium containing 0.1% FBS in the presence of Dyn2 inhibitors or DMSO as indicated. Representative images of inhibitor-treated and control cells (stained with Oil Red O) are shown in A and B together with the quantitation of the average LD area per cell from three independent experiments. Pharmacological inhibitors used were: Dynasore (inhibits Dyn2 GTPase activity), MiTMAB (targets PH domain and interferes with membrane binding), Dynole 34-2 (allosteric GTPase inhibitor), and Dynole 31-2 (negative control for Dynole 34-2). Bars, 20 µM. (C) Representative images from control and Dyn2 knockout MEFs after an overnight lipid loading with 400 µM oleate for 17 h. Knockout of Dyn2 was induced by treatment with 2 µM 4-hydroxy-tamoxifen for 7 d and was confirmed by immunostaining of endogenous Dyn2 (top row) and by immunoblot (D). Bars, 20 µM. (E and F) Average LD number (E) and area (F) per cell from five independent experiments. All data are represented as mean ± SE. *, P ≤ 0.05; **, P ≤ 0.01. (G) Whole-cell lysates and LD fractions isolated from HuH-7 hepatocytes under resting or starved (2 h HBSS starvation) conditions. (H) Primary hepatocyte expressing Dyn2-GFP, showing an absence of colocalization with the LD surface (stained with Oil Red O). Bar, 20 µM. Inset shows magnification of boxed region (bar, 2 µM).

    Journal: The Journal of Cell Biology

    Article Title: Lipid droplet breakdown requires Dynamin 2 for vesiculation of autolysosomal tubules in hepatocytes

    doi: 10.1083/jcb.201306140

    Figure Lengend Snippet: Pharmacological and genetic inhibition of Dyn2 function also reduces starvation-induced LD breakdown. HuH-7 (A) and Hep3B (B) hepatocytes were loaded with 150 µM oleate overnight and starved for 48 h in medium containing 0.1% FBS in the presence of Dyn2 inhibitors or DMSO as indicated. Representative images of inhibitor-treated and control cells (stained with Oil Red O) are shown in A and B together with the quantitation of the average LD area per cell from three independent experiments. Pharmacological inhibitors used were: Dynasore (inhibits Dyn2 GTPase activity), MiTMAB (targets PH domain and interferes with membrane binding), Dynole 34-2 (allosteric GTPase inhibitor), and Dynole 31-2 (negative control for Dynole 34-2). Bars, 20 µM. (C) Representative images from control and Dyn2 knockout MEFs after an overnight lipid loading with 400 µM oleate for 17 h. Knockout of Dyn2 was induced by treatment with 2 µM 4-hydroxy-tamoxifen for 7 d and was confirmed by immunostaining of endogenous Dyn2 (top row) and by immunoblot (D). Bars, 20 µM. (E and F) Average LD number (E) and area (F) per cell from five independent experiments. All data are represented as mean ± SE. *, P ≤ 0.05; **, P ≤ 0.01. (G) Whole-cell lysates and LD fractions isolated from HuH-7 hepatocytes under resting or starved (2 h HBSS starvation) conditions. (H) Primary hepatocyte expressing Dyn2-GFP, showing an absence of colocalization with the LD surface (stained with Oil Red O). Bar, 20 µM. Inset shows magnification of boxed region (bar, 2 µM).

    Article Snippet: The Dynasore and MiTMAB inhibitory compounds were from EMD Millipore and the Dynole series of compounds were from Abcam.

    Techniques: Inhibition, Staining, Quantitation Assay, Activity Assay, Binding Assay, Negative Control, Knock-Out, Immunostaining, Isolation, Expressing

    Dyn2 associates with autolysosomal tubules. (A and B) Subcellular density gradient fractionation of Hep3B hepatocytes starved for 2 h in HBSS and treated with 40 µM Dynasore, to induce tubule formation, as in Fig. 5 . Cells were lysed (WCL), and the post-nuclear supernatant (PNS) was pelleted by centrifugation to produce a crude lysosomal fraction (CLF) and high-speed supernatant (HSS). The CLF was subsequently loaded onto an 8–27% discontinuous iodixanol (OptiPrep) gradient for separation by ultracentrifugation. Nine fractions were collected from the top of the gradient and blotted for Dyn2 and the lysosomal resident protein, LAMP1. Lysosomal acid phosphatase activity roughly correlates with LAMP1 protein levels in each fraction. Levels of Dyn2 are highest in fraction 2, corresponding with both the peak levels of LAMP1 and lysosomal activity. (A) The data shown are from a single representative experiment out of three repeats. (B) Blotting for subcellular components shows that Dyn2 is specific for these same fractions, unlike other organelle markers such as EEA1 (early endosomes) and COXIV (mitochondria). (C and D) Dyn2 localizes to the surface of LAMP1-positive compartments. Fluorescence imaging of Hep3B hepatocytes transfected with Dyn2-GFP and LAMP1-mCherry under resting (C and C′) or starvation (D) conditions. (C′′) Dyn2-GFP localizing to LAMP1-labeled lysosome structures under starvation conditions. Arrows indicate regions of protein colocalization. Dyn2 (arrows) is present at the site of scission of LAMP1-positive tubules (arrowhead) from large autolysosomal structures. Bars: (C) 5 µM; (C′, C′′, and D) 3 µM.

    Journal: The Journal of Cell Biology

    Article Title: Lipid droplet breakdown requires Dynamin 2 for vesiculation of autolysosomal tubules in hepatocytes

    doi: 10.1083/jcb.201306140

    Figure Lengend Snippet: Dyn2 associates with autolysosomal tubules. (A and B) Subcellular density gradient fractionation of Hep3B hepatocytes starved for 2 h in HBSS and treated with 40 µM Dynasore, to induce tubule formation, as in Fig. 5 . Cells were lysed (WCL), and the post-nuclear supernatant (PNS) was pelleted by centrifugation to produce a crude lysosomal fraction (CLF) and high-speed supernatant (HSS). The CLF was subsequently loaded onto an 8–27% discontinuous iodixanol (OptiPrep) gradient for separation by ultracentrifugation. Nine fractions were collected from the top of the gradient and blotted for Dyn2 and the lysosomal resident protein, LAMP1. Lysosomal acid phosphatase activity roughly correlates with LAMP1 protein levels in each fraction. Levels of Dyn2 are highest in fraction 2, corresponding with both the peak levels of LAMP1 and lysosomal activity. (A) The data shown are from a single representative experiment out of three repeats. (B) Blotting for subcellular components shows that Dyn2 is specific for these same fractions, unlike other organelle markers such as EEA1 (early endosomes) and COXIV (mitochondria). (C and D) Dyn2 localizes to the surface of LAMP1-positive compartments. Fluorescence imaging of Hep3B hepatocytes transfected with Dyn2-GFP and LAMP1-mCherry under resting (C and C′) or starvation (D) conditions. (C′′) Dyn2-GFP localizing to LAMP1-labeled lysosome structures under starvation conditions. Arrows indicate regions of protein colocalization. Dyn2 (arrows) is present at the site of scission of LAMP1-positive tubules (arrowhead) from large autolysosomal structures. Bars: (C) 5 µM; (C′, C′′, and D) 3 µM.

    Article Snippet: The Dynasore and MiTMAB inhibitory compounds were from EMD Millipore and the Dynole series of compounds were from Abcam.

    Techniques: Fractionation, Centrifugation, Activity Assay, Fluorescence, Imaging, Transfection, Labeling

    Dyn2 inhibition leads to enlarged autolysosomal structures and prevents the autophagy of lipid droplets. Hep3B cells treated with either a nontargeting control siRNA (A and B, siNT) or an siRNA targeting human Dyn2 (C and D, siDyn2) were fixed and co-stained with antibodies specific for LAMP1 (red) and LC3 (green). After Dyn2 knockdown, a juxtanuclear aggregation and enlargement of the LAMP1-positive compartment is observed (C and D, arrows). Increased labeling of LC3 is also detectable after knockdown of Dyn2. (E) Western blotting of Hep3B lysates after a 3-d treatment with either the control or Dyn2-targeted siRNA and further treatment with or without 50 µM leupeptin. Densitometry-based analysis of six independent experiments is shown at the bottom of the figure. (F) Western blotting of Hep3B lysates after treatment for 2 h with DMSO or 80 µM Dynasore, in the presence or absence of 50 µM leupeptin. Quantitation of LC3-II levels relative to control are shown below the blots. The data are represented as mean ± SE; *, P ≤ 0.05.

    Journal: The Journal of Cell Biology

    Article Title: Lipid droplet breakdown requires Dynamin 2 for vesiculation of autolysosomal tubules in hepatocytes

    doi: 10.1083/jcb.201306140

    Figure Lengend Snippet: Dyn2 inhibition leads to enlarged autolysosomal structures and prevents the autophagy of lipid droplets. Hep3B cells treated with either a nontargeting control siRNA (A and B, siNT) or an siRNA targeting human Dyn2 (C and D, siDyn2) were fixed and co-stained with antibodies specific for LAMP1 (red) and LC3 (green). After Dyn2 knockdown, a juxtanuclear aggregation and enlargement of the LAMP1-positive compartment is observed (C and D, arrows). Increased labeling of LC3 is also detectable after knockdown of Dyn2. (E) Western blotting of Hep3B lysates after a 3-d treatment with either the control or Dyn2-targeted siRNA and further treatment with or without 50 µM leupeptin. Densitometry-based analysis of six independent experiments is shown at the bottom of the figure. (F) Western blotting of Hep3B lysates after treatment for 2 h with DMSO or 80 µM Dynasore, in the presence or absence of 50 µM leupeptin. Quantitation of LC3-II levels relative to control are shown below the blots. The data are represented as mean ± SE; *, P ≤ 0.05.

    Article Snippet: The Dynasore and MiTMAB inhibitory compounds were from EMD Millipore and the Dynole series of compounds were from Abcam.

    Techniques: Inhibition, Staining, Labeling, Western Blot, Quantitation Assay

    Acute inhibition of Dyn2 reversibly disrupts autophagic lysosomal reformation (ALR) and lysosomal tubule scission. (A–D) Still frames from time-lapse movies of Hep3B cells expressing LAMP1-mCherry. Cells were starved for 2 h in HBSS and subsequently treated for 30 min with either DMSO (A and B) or 40 µM Dynasore (C and D), which induced extensive tubulation of LAMP1-positive compartments. Bars (A–D): 20 µM; (A′–B′) 2 µM; (C′–D′) 10 µM. (E–G) To demonstrate the reversibility of this tubulation, Dynasore-treated cells were washed extensively with drug-free media containing 10% FBS and monitored by time-lapse microscopy for 45 min. Frequently, after drug washout, LAMP1-positive tubules exhibited noticeable varicosities (E and F, arrows; bars, 10 µM) along their length. These sites are suggestive of areas of scission and resumed budding of nascent protolysosomes from the reformation tubules (G; bars, 10 µM). (H) Tubules from cells undergoing drug washout were quantified by tracing their lengths at the beginning and end of these movies. Still frames from a representative movie show tubule content at t = 10 and 45 min after drug washout. Bars, 20 µM. (I) Analysis of five independent movies showed an average decrease in total tubulation of ∼50% after drug washout. Data represent the average relative change in total tubule length between the first and last frames of the time-lapse movies. Error bars represent SE; *, P

    Journal: The Journal of Cell Biology

    Article Title: Lipid droplet breakdown requires Dynamin 2 for vesiculation of autolysosomal tubules in hepatocytes

    doi: 10.1083/jcb.201306140

    Figure Lengend Snippet: Acute inhibition of Dyn2 reversibly disrupts autophagic lysosomal reformation (ALR) and lysosomal tubule scission. (A–D) Still frames from time-lapse movies of Hep3B cells expressing LAMP1-mCherry. Cells were starved for 2 h in HBSS and subsequently treated for 30 min with either DMSO (A and B) or 40 µM Dynasore (C and D), which induced extensive tubulation of LAMP1-positive compartments. Bars (A–D): 20 µM; (A′–B′) 2 µM; (C′–D′) 10 µM. (E–G) To demonstrate the reversibility of this tubulation, Dynasore-treated cells were washed extensively with drug-free media containing 10% FBS and monitored by time-lapse microscopy for 45 min. Frequently, after drug washout, LAMP1-positive tubules exhibited noticeable varicosities (E and F, arrows; bars, 10 µM) along their length. These sites are suggestive of areas of scission and resumed budding of nascent protolysosomes from the reformation tubules (G; bars, 10 µM). (H) Tubules from cells undergoing drug washout were quantified by tracing their lengths at the beginning and end of these movies. Still frames from a representative movie show tubule content at t = 10 and 45 min after drug washout. Bars, 20 µM. (I) Analysis of five independent movies showed an average decrease in total tubulation of ∼50% after drug washout. Data represent the average relative change in total tubule length between the first and last frames of the time-lapse movies. Error bars represent SE; *, P

    Article Snippet: The Dynasore and MiTMAB inhibitory compounds were from EMD Millipore and the Dynole series of compounds were from Abcam.

    Techniques: Inhibition, Expressing, Time-lapse Microscopy

    CVB3-RD entry requires dynamin. CVB3-RD particles were bound to and allowed to enter cells pretreated with DMSO or 200 μM dynasore. Unbound virus was washed off, complete medium with NuSerum and dynasore was added, and virus was allowed to enter

    Journal:

    Article Title: Dynamin- and Lipid Raft-Dependent Entry of Decay-Accelerating Factor (DAF)-Binding and Non-DAF-Binding Coxsackieviruses into Nonpolarized Cells ▿

    doi: 10.1128/JVI.01016-09

    Figure Lengend Snippet: CVB3-RD entry requires dynamin. CVB3-RD particles were bound to and allowed to enter cells pretreated with DMSO or 200 μM dynasore. Unbound virus was washed off, complete medium with NuSerum and dynasore was added, and virus was allowed to enter

    Article Snippet: Dynasore, genistein, PP2, and bafilomycin A1 were obtained from Calbiochem (La Jolla, CA); chlorpromazine (CPZ), filipin III, NH4 Cl, methyl-beta-cyclodextrin (MβCD), and water-soluble cholesterol from Sigma (St. Louis, MO); and imatinib from LC Laboratories (Woburn, MA).

    Techniques:

    Dynasore inhibits wound edge actin remodeling by mammalian cells. Confluent monolayers of mIMCD3 mouse kidney epithelial cells were scratch wounded after treatment with DMSO (control) or 80 µM dynasore, a chemical inhibitor of Dynamin. At the indicated time points, the cells were fixed and stained with rhodamine-phalloidin and antibodies against phospho (=activated) myosin light chain (pMLC) or clathrin heavy chain (CHC), to examine the formation of an actomyosin cable and actin protrusions at the wound edge and the intracellular localization of clathrin. (A) Representative phalloidin and anti-pMLC images of control and dynasore-treated cells at 0 and 15 min after wounding. Arrows and arrowheads indicate the actin (myosin) cable and protrusions, respectively. (B) Cable and protrusion formation in the images 15 min after wounding was quantified. Cable formation was quantified using both the F-actin and pMLC images. Protrusions were quantified using the F-actin images. Note that the formation of both actin cable and protrusions are inhibited by dynasore. (C) Low magnification phalloidin images at 0 and 4 h after wounding. Note that the advancement of the cell sheets is inhibited by dynasore. At 4 h, a wound edge actin cable is observed, even for dynasore-treated cell sheets. (D) Quantification of wound width in each sample at the indicated time points. (E) Representative phalloidin and anti–clathrin heavy chain images of control and dynasore-treated cells at 15 min after wounding. Bars in the column scatter plots indicate means ± SEM of all plotted values. Line graphs show means ± SEM of the data. Bars: (A and E) 20 µm; (C) 100 µm.

    Journal: The Journal of Cell Biology

    Article Title: Endocytosis-dependent coordination of multiple actin regulators is required for wound healing

    doi: 10.1083/jcb.201411037

    Figure Lengend Snippet: Dynasore inhibits wound edge actin remodeling by mammalian cells. Confluent monolayers of mIMCD3 mouse kidney epithelial cells were scratch wounded after treatment with DMSO (control) or 80 µM dynasore, a chemical inhibitor of Dynamin. At the indicated time points, the cells were fixed and stained with rhodamine-phalloidin and antibodies against phospho (=activated) myosin light chain (pMLC) or clathrin heavy chain (CHC), to examine the formation of an actomyosin cable and actin protrusions at the wound edge and the intracellular localization of clathrin. (A) Representative phalloidin and anti-pMLC images of control and dynasore-treated cells at 0 and 15 min after wounding. Arrows and arrowheads indicate the actin (myosin) cable and protrusions, respectively. (B) Cable and protrusion formation in the images 15 min after wounding was quantified. Cable formation was quantified using both the F-actin and pMLC images. Protrusions were quantified using the F-actin images. Note that the formation of both actin cable and protrusions are inhibited by dynasore. (C) Low magnification phalloidin images at 0 and 4 h after wounding. Note that the advancement of the cell sheets is inhibited by dynasore. At 4 h, a wound edge actin cable is observed, even for dynasore-treated cell sheets. (D) Quantification of wound width in each sample at the indicated time points. (E) Representative phalloidin and anti–clathrin heavy chain images of control and dynasore-treated cells at 15 min after wounding. Bars in the column scatter plots indicate means ± SEM of all plotted values. Line graphs show means ± SEM of the data. Bars: (A and E) 20 µm; (C) 100 µm.

    Article Snippet: Dynasore (EMD Millipore) was diluted to 100 mM stock in DMSO.

    Techniques: Staining