Structured Review

Millipore dynasore
USP47 knockdown inhibits influenza virus entry. ( a ) Fluorescence detection of GFP insert in cells under various experimental conditions. ( b ) Virus entry was determined by flow cytometry with permeabilized (intracellular) and nonpermeabilized (cell surface) USP47 knockdown cells infected with NY55 at an MOI of 50. Virus entry was determined by detection with anti-NP mouse monoclonal antibody conjugated with Alexa Fluor 350. <t>Dynasore,</t> a known inhibitor of influenza virus entry, was used as a positive control. shNSi indicates shRNA nontargeting control. ( c ) Quantitation of the two upper quandrants (+/− and ++) after each condition. Data are representative of three replicates
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1) Product Images from "Knockdown of specific host factors protects against influenza virus-induced cell death"

Article Title: Knockdown of specific host factors protects against influenza virus-induced cell death

Journal: Cell Death & Disease

doi: 10.1038/cddis.2013.296

USP47 knockdown inhibits influenza virus entry. ( a ) Fluorescence detection of GFP insert in cells under various experimental conditions. ( b ) Virus entry was determined by flow cytometry with permeabilized (intracellular) and nonpermeabilized (cell surface) USP47 knockdown cells infected with NY55 at an MOI of 50. Virus entry was determined by detection with anti-NP mouse monoclonal antibody conjugated with Alexa Fluor 350. Dynasore, a known inhibitor of influenza virus entry, was used as a positive control. shNSi indicates shRNA nontargeting control. ( c ) Quantitation of the two upper quandrants (+/− and ++) after each condition. Data are representative of three replicates
Figure Legend Snippet: USP47 knockdown inhibits influenza virus entry. ( a ) Fluorescence detection of GFP insert in cells under various experimental conditions. ( b ) Virus entry was determined by flow cytometry with permeabilized (intracellular) and nonpermeabilized (cell surface) USP47 knockdown cells infected with NY55 at an MOI of 50. Virus entry was determined by detection with anti-NP mouse monoclonal antibody conjugated with Alexa Fluor 350. Dynasore, a known inhibitor of influenza virus entry, was used as a positive control. shNSi indicates shRNA nontargeting control. ( c ) Quantitation of the two upper quandrants (+/− and ++) after each condition. Data are representative of three replicates

Techniques Used: Fluorescence, Flow Cytometry, Cytometry, Infection, Positive Control, shRNA, Quantitation Assay

2) Product Images from "CAR regulates epithelial cell junction stability through control of E-cadherin trafficking"

Article Title: CAR regulates epithelial cell junction stability through control of E-cadherin trafficking

Journal: Scientific Reports

doi: 10.1038/srep02889

CAR promotes endocytosis through a macropinocytosis-like, dynamic dependent mechanism. (A) Confocal microscopy of endocytosing E-cadherin in WT and CARGFP HBEC in HECD-1 antibody internalisation assay. Arrows highlight large E-cadherin positive vesicles. (B) Quantification of large, macropinosome-like E-cadherin containing structures observed in HECD-1 internalisation assay after 60 min of internalisation. Cells with large E-cadherin containing vesicles were counted and presented as a percentage. Images are maximum intensity projections of Z-stacks. (C) Confocal microscopy of endocytosing E-cadherin in WT and CARGFP HBEC in HECD-1 antibody internalisation assay, either untreated or treated with 100 μM EIPA for 30 min prior to addition of HECD-1 and during assay. (D) Confocal microscopy of E-cadherin in 50:50 mix of WT and CARGFP HBEC, either untreated or treated with 100 μM EIPA for 30 min prior to fixation. (E) Confocal microscopy of E-cadherin in DMSO control or Dynasore treated WT and CARGFP HBEC. (F) Confocal microscopy of endocytosing E-cadherin in WT and CARGFP HBEC in HECD-1 antibody internalisation assay. Cells were fixed after 60 min of HECD-1 internalisation and co-stained with anti-Rabankyrin antibody (H) or anti-LAMP1 antibody. Colocalisation is shown in yellow. (G) Confocal microscopy of endocytosing E-cadherin in CARGFP HBEC in HECD-1 internalisation assay performed in the presence of Dextran-597, and fixed after 60 min of HECD-1 internalisation. Arrow highlights colocalisation between internalised Dextran-597 and internalised HECD-1 which is shown in yellow. (I) Confocal microscopy of endocytosing E-cadherin, in Rab7-RFP expressing CARGFP HBEC, in HECD-1 antibody internalisation assay. Cells were fixed after 60 min of HECD-1 internalisation. Colocalisation between Rab7-RFP and HECD-1 is shown in yellow. Scale bars correspond to 10 μm.
Figure Legend Snippet: CAR promotes endocytosis through a macropinocytosis-like, dynamic dependent mechanism. (A) Confocal microscopy of endocytosing E-cadherin in WT and CARGFP HBEC in HECD-1 antibody internalisation assay. Arrows highlight large E-cadherin positive vesicles. (B) Quantification of large, macropinosome-like E-cadherin containing structures observed in HECD-1 internalisation assay after 60 min of internalisation. Cells with large E-cadherin containing vesicles were counted and presented as a percentage. Images are maximum intensity projections of Z-stacks. (C) Confocal microscopy of endocytosing E-cadherin in WT and CARGFP HBEC in HECD-1 antibody internalisation assay, either untreated or treated with 100 μM EIPA for 30 min prior to addition of HECD-1 and during assay. (D) Confocal microscopy of E-cadherin in 50:50 mix of WT and CARGFP HBEC, either untreated or treated with 100 μM EIPA for 30 min prior to fixation. (E) Confocal microscopy of E-cadherin in DMSO control or Dynasore treated WT and CARGFP HBEC. (F) Confocal microscopy of endocytosing E-cadherin in WT and CARGFP HBEC in HECD-1 antibody internalisation assay. Cells were fixed after 60 min of HECD-1 internalisation and co-stained with anti-Rabankyrin antibody (H) or anti-LAMP1 antibody. Colocalisation is shown in yellow. (G) Confocal microscopy of endocytosing E-cadherin in CARGFP HBEC in HECD-1 internalisation assay performed in the presence of Dextran-597, and fixed after 60 min of HECD-1 internalisation. Arrow highlights colocalisation between internalised Dextran-597 and internalised HECD-1 which is shown in yellow. (I) Confocal microscopy of endocytosing E-cadherin, in Rab7-RFP expressing CARGFP HBEC, in HECD-1 antibody internalisation assay. Cells were fixed after 60 min of HECD-1 internalisation. Colocalisation between Rab7-RFP and HECD-1 is shown in yellow. Scale bars correspond to 10 μm.

Techniques Used: Confocal Microscopy, Internalisation Assay, Staining, Expressing

3) 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

4) 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

5) 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

6) 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

7) 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

8) 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

9) Product Images from "Hippocampal Long-Term Depression in the Presence of Calcium-Permeable AMPA Receptors"

Article Title: Hippocampal Long-Term Depression in the Presence of Calcium-Permeable AMPA Receptors

Journal: Frontiers in Synaptic Neuroscience

doi: 10.3389/fnsyn.2018.00041

mGluR-LTD in mature GluA2 KO mice. (A) pep4c has no effect on either basal synaptic responses or PP-LFS induced LTD in mature GluA2 KO mice. (B) pep2m has no effect on basal synaptic responses, but rescues LTD induced by PP-LFS in mature GluA2 KO mice. Dynasore (80 μM) blocks PP-LFS induced LTD in WT mice. (C) S3 but not pS3 rescues PP-LFS induced LTD in mature GluA2 KO mice. (D) S3 but not pS3 rescues DHPG-induced LTD in mature GluA2 KO mice.
Figure Legend Snippet: mGluR-LTD in mature GluA2 KO mice. (A) pep4c has no effect on either basal synaptic responses or PP-LFS induced LTD in mature GluA2 KO mice. (B) pep2m has no effect on basal synaptic responses, but rescues LTD induced by PP-LFS in mature GluA2 KO mice. Dynasore (80 μM) blocks PP-LFS induced LTD in WT mice. (C) S3 but not pS3 rescues PP-LFS induced LTD in mature GluA2 KO mice. (D) S3 but not pS3 rescues DHPG-induced LTD in mature GluA2 KO mice.

Techniques Used: Mouse Assay

10) Product Images from "Endocytosis-dependent coordination of multiple actin regulators is required for wound healing"

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

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201411037

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.
Figure Legend 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.

Techniques Used: Staining

11) Product Images from "Assessing mechanisms of GPIHBP1 and lipoprotein lipase movement across endothelial cells [S]"

Article Title: Assessing mechanisms of GPIHBP1 and lipoprotein lipase movement across endothelial cells [S]

Journal: Journal of Lipid Research

doi: 10.1194/jlr.M031559

Inhibition of LPL transport across GPIHBP1-expressing endothelial cells grown on transwell filters by dynasore (A) and genistein (B). Bar graphs show the fold change (normalized to vehicle-treated CD59-expressing control cells; mean ± SEM;) in
Figure Legend Snippet: Inhibition of LPL transport across GPIHBP1-expressing endothelial cells grown on transwell filters by dynasore (A) and genistein (B). Bar graphs show the fold change (normalized to vehicle-treated CD59-expressing control cells; mean ± SEM;) in

Techniques Used: Inhibition, Expressing

12) Product Images from "Hsp70 downregulates dynamin in experimental stroke: a new therapeutic target?"

Article Title: Hsp70 downregulates dynamin in experimental stroke: a new therapeutic target?

Journal: Stroke; a journal of cerebral circulation

doi: 10.1161/STROKEAHA.116.012763

Dynamin inhibition is neuroprotective. Cultures of N2a cells were subjected to OGD and treated with dynasore (Dyna) and vehicle (Veh), a dynamin inhibitor. MTT (A) and trypan blue stains (B) showed increased cell viability and decreased cell death, respectively
Figure Legend Snippet: Dynamin inhibition is neuroprotective. Cultures of N2a cells were subjected to OGD and treated with dynasore (Dyna) and vehicle (Veh), a dynamin inhibitor. MTT (A) and trypan blue stains (B) showed increased cell viability and decreased cell death, respectively

Techniques Used: Inhibition, MTT Assay

13) Product Images from "Fusion of Anthopleurin-B to AAV2 increases specificity of cardiac gene transfer"

Article Title: Fusion of Anthopleurin-B to AAV2 increases specificity of cardiac gene transfer

Journal: Virology

doi: 10.1016/j.virol.2017.10.006

Attachment of AAV and AAV-ApB vectors Attachment efficiency to (a) isolated and immortalized cardiac myocytes, and (b) non-cardiac cells with and without expression of the cardiac sodium channel (Nav1.5) was evaluated by DNA quantification of dsRed, normalized to 28S. Cells were first chilled with PBS, and pretreated with Dynasore to prevent internalization of particles after virus exposure. Cells were exposed to virus for 60 minutes, subsequently incubated at 4 °C for 5 minutes, and washed three times to detach loosely bound virus; cells were then harvested by scraping (n=3 per data point). Attachment of HA-AAV-ApB is superior to that of HA-AAV in both immortalized and isolated cardiomyocytes (p
Figure Legend Snippet: Attachment of AAV and AAV-ApB vectors Attachment efficiency to (a) isolated and immortalized cardiac myocytes, and (b) non-cardiac cells with and without expression of the cardiac sodium channel (Nav1.5) was evaluated by DNA quantification of dsRed, normalized to 28S. Cells were first chilled with PBS, and pretreated with Dynasore to prevent internalization of particles after virus exposure. Cells were exposed to virus for 60 minutes, subsequently incubated at 4 °C for 5 minutes, and washed three times to detach loosely bound virus; cells were then harvested by scraping (n=3 per data point). Attachment of HA-AAV-ApB is superior to that of HA-AAV in both immortalized and isolated cardiomyocytes (p

Techniques Used: Isolation, Expressing, Incubation

14) Product Images from "Maintenance of CD4 T cell fitness through regulation of Foxo1"

Article Title: Maintenance of CD4 T cell fitness through regulation of Foxo1

Journal: Nature immunology

doi: 10.1038/s41590-018-0157-4

Impaired lysosomal biogenesis and endocytosis of IL-2Rβ upon maintaining Foxo1 activity. a , Immunoblot analysis of isocultures stimulated for 1 or 2 d with plate-bound anti-CD3 and soluble anti-CD28. After sorting, a proportion of GFP – and GFP + cells were immediately lysed (day 0). Data are representative of four independent experiments with similar results. β-actin was used as a loading control. b , Flow cytometric analysis and quantification of isocultures stimulated for 1 or 2 d with plate-bound anti-CD3 and soluble anti-CD28 with vehicle (Veh) or Dynasore (Dyna; 80 and 140 mM, Dyna80 and Dyna140, respectively; n = 5). Representative plots are of day 1 post-activation with 140 mM Dynasore. Quantification involved a two-tailed Student’s t -test with no adjustments made for multiple comparisons; center value, mean; error bars, s.d. * P
Figure Legend Snippet: Impaired lysosomal biogenesis and endocytosis of IL-2Rβ upon maintaining Foxo1 activity. a , Immunoblot analysis of isocultures stimulated for 1 or 2 d with plate-bound anti-CD3 and soluble anti-CD28. After sorting, a proportion of GFP – and GFP + cells were immediately lysed (day 0). Data are representative of four independent experiments with similar results. β-actin was used as a loading control. b , Flow cytometric analysis and quantification of isocultures stimulated for 1 or 2 d with plate-bound anti-CD3 and soluble anti-CD28 with vehicle (Veh) or Dynasore (Dyna; 80 and 140 mM, Dyna80 and Dyna140, respectively; n = 5). Representative plots are of day 1 post-activation with 140 mM Dynasore. Quantification involved a two-tailed Student’s t -test with no adjustments made for multiple comparisons; center value, mean; error bars, s.d. * P

Techniques Used: Activity Assay, Flow Cytometry, Activation Assay, Two Tailed Test

15) Product Images from "Notch Promotes Dynamin-Dependent Endocytosis of Nephrin"

Article Title: Notch Promotes Dynamin-Dependent Endocytosis of Nephrin

Journal: Journal of the American Society of Nephrology : JASN

doi: 10.1681/ASN.2011010027

Notch-IC activity promotes nephrin internalization through a dynamin-dependent, raft-independent process. Shown are representative, merged images of HEK293T cells stained with anti-nephrin (green), anti-FLAG (red), and WGA (blue) to detect Nephrin MYC , NOTCH1-IC FLAG , and cell membrane, respectively; original magnification, ×600. Cells were preincubated with either DMSO vehicle (A, A', D, and D'), Dynasore 160 μM (C, C', D, and D'), or M β CD 10 mM (E, E', F, and F'). (A, C, and E) Multichannel merged images depicting nephrin staining in Nephrin MYC -expressing HEK293T cells. (B, D, and F) Multichannel merged images of nephrin and Notch-IC FLAG staining in HEK293T cells expressing both Nephrin MYC and NOTCH1-IC FLAG . (A'–F') Enlarged view of areas represented by white boxes in corresponding panels A–F. Nuclei were counterstained with 4′-6-diamidino-2-phenylindole (recolorized as gray). (A, C, and E) Nephrin staining co-localizes with WGA along the peripheral margins of cells expressing Nephrin MYC alone (A and A') and is unaffected by preincubation with Dynasore (C and C'). M β CD pretreatment resulted in a small increase in cytoplasmic staining for nephrin in cells expressing Nephrin MYC alone (E and E') compared with cells preincubated with vehicle (A and A'). (B, D, and F) In the presence of vehicle alone (B), nephrin staining is strongly detected within the cytoplasm of cells, which also show nuclear staining for NOTCH1-IC FLAG (B', white arrow). Pretreatment with Dynasore (D) decreased the amount of cytoplasmic nephrin staining in NOTCH1-IC FLAG –expressing cells (D'), whereas M β CD did not block nephrin internalization (F) as revealed by comparable patterns of anti-nephrin staining within the cytoplasm of NOTCH1-IC FLAG –expressing cells (red nuclei) in both the absence (B') and presence (F') of M β CD. (G and H) Histograms showing the effect of Dynasore and M β CD on nephrin cell-surface localization as quantified by anti-nephrin immunofluorescence intensity in Nephrin MYC -expressing cells (G) and in cells expressing both Nephrin MYC and NOTCH1-IC FLAG (H). Shown are mean values of cell surface–associated nephrin calculated as the difference between total and cytoplasmic anti-nephrin immunofluorescence intensities in single cells and expressed as a percentage of total cell anti-nephrin immunofluorescence intensity. Error bars denote SEM. Experiments were performed in duplicate. White bars, DMSO treated; (G), n =28 cells; (H), n =20 cells. Gray bars, Dynasore treated; (G), n =25 cells; (H), n =22 cells. Black bars, M β CD treated; (G), n =18 cells; (H), n =15 cells.
Figure Legend Snippet: Notch-IC activity promotes nephrin internalization through a dynamin-dependent, raft-independent process. Shown are representative, merged images of HEK293T cells stained with anti-nephrin (green), anti-FLAG (red), and WGA (blue) to detect Nephrin MYC , NOTCH1-IC FLAG , and cell membrane, respectively; original magnification, ×600. Cells were preincubated with either DMSO vehicle (A, A', D, and D'), Dynasore 160 μM (C, C', D, and D'), or M β CD 10 mM (E, E', F, and F'). (A, C, and E) Multichannel merged images depicting nephrin staining in Nephrin MYC -expressing HEK293T cells. (B, D, and F) Multichannel merged images of nephrin and Notch-IC FLAG staining in HEK293T cells expressing both Nephrin MYC and NOTCH1-IC FLAG . (A'–F') Enlarged view of areas represented by white boxes in corresponding panels A–F. Nuclei were counterstained with 4′-6-diamidino-2-phenylindole (recolorized as gray). (A, C, and E) Nephrin staining co-localizes with WGA along the peripheral margins of cells expressing Nephrin MYC alone (A and A') and is unaffected by preincubation with Dynasore (C and C'). M β CD pretreatment resulted in a small increase in cytoplasmic staining for nephrin in cells expressing Nephrin MYC alone (E and E') compared with cells preincubated with vehicle (A and A'). (B, D, and F) In the presence of vehicle alone (B), nephrin staining is strongly detected within the cytoplasm of cells, which also show nuclear staining for NOTCH1-IC FLAG (B', white arrow). Pretreatment with Dynasore (D) decreased the amount of cytoplasmic nephrin staining in NOTCH1-IC FLAG –expressing cells (D'), whereas M β CD did not block nephrin internalization (F) as revealed by comparable patterns of anti-nephrin staining within the cytoplasm of NOTCH1-IC FLAG –expressing cells (red nuclei) in both the absence (B') and presence (F') of M β CD. (G and H) Histograms showing the effect of Dynasore and M β CD on nephrin cell-surface localization as quantified by anti-nephrin immunofluorescence intensity in Nephrin MYC -expressing cells (G) and in cells expressing both Nephrin MYC and NOTCH1-IC FLAG (H). Shown are mean values of cell surface–associated nephrin calculated as the difference between total and cytoplasmic anti-nephrin immunofluorescence intensities in single cells and expressed as a percentage of total cell anti-nephrin immunofluorescence intensity. Error bars denote SEM. Experiments were performed in duplicate. White bars, DMSO treated; (G), n =28 cells; (H), n =20 cells. Gray bars, Dynasore treated; (G), n =25 cells; (H), n =22 cells. Black bars, M β CD treated; (G), n =18 cells; (H), n =15 cells.

Techniques Used: Activity Assay, Staining, Whole Genome Amplification, Expressing, Blocking Assay, Immunofluorescence

16) Product Images from "Acute dynamin inhibition dissects synaptic vesicle recycling pathways that drive spontaneous and evoked neurotransmission"

Article Title: Acute dynamin inhibition dissects synaptic vesicle recycling pathways that drive spontaneous and evoked neurotransmission

Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

doi: 10.1523/JNEUROSCI.3427-09.2010

Spontaneous and evoked transmission occur at the same synapses and are differentially affected by dynasore
Figure Legend Snippet: Spontaneous and evoked transmission occur at the same synapses and are differentially affected by dynasore

Techniques Used: Transmission Assay

Incubation with dynasore during stimulation causes vesicle depletion
Figure Legend Snippet: Incubation with dynasore during stimulation causes vesicle depletion

Techniques Used: Incubation

Prolonged dynasore application failed to suppress spontaneous neurotransmission
Figure Legend Snippet: Prolonged dynasore application failed to suppress spontaneous neurotransmission

Techniques Used:

17) Product Images from "Type I Interferon Production Induced by Streptococcus pyogenes-Derived Nucleic Acids Is Required for Host Protection"

Article Title: Type I Interferon Production Induced by Streptococcus pyogenes-Derived Nucleic Acids Is Required for Host Protection

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1001345

IFN-β production in response to S. pyogenes depends on phagosome maturation. ( A ) BMDMs pretreated (for 45 min) with dynasore or left untreated were infected with S. pyogenes (MOI = 100) for the indicated periods or left uninfected. At the indicated time points total RNA was isolated, reverse-transcribed and analyzed by qPCR for IFN-β. HPRT was used for normalization. Values represent mean ± SD; n = 3. ( B ) BMDMs pretreated (for 45 min) with dynasore or left untreated were infected with CFSE-labeled (green) S. pyogenes (MOI = 100) and fixed at indicated time points. Extracellular S. pyogenes were stained with anti- S. pyogenes antibody (red). The percentages of internalized S. pyogenes were calculated based on the number of red and green fluorescing bacteria in a field. At least five fields were counted and SDs were calculated. ( C ) Fluorescence microscopy imaging of cells treated as described in (B). Total S. pyogenes labeled with CFSE (green) and extracellular S. pyogenes stained post-fixation with anti- S. pyogenes antibody (red), as well as merge are shown. Green cells in the merge image represent internalized bacteria. Representative panels are shown. ( D ) BMDMs pretreated (for 45 min) with bafilomycin A1 or left untreated were infected with S. pyogenes (MOI = 100). At indicated time points, supernatants were collected and IFN-β release was measured as described in (A). Values represent mean ± SD; n = 3. ( E ) BMDMs treated with dynasore, bafilomycin A or left untreated were infected with S. pyogenes (MOI = 100). At indicated time points, serial dilutions of cellular lysates were plated to count colony-forming units (CFUs) that are displayed as log CFU.
Figure Legend Snippet: IFN-β production in response to S. pyogenes depends on phagosome maturation. ( A ) BMDMs pretreated (for 45 min) with dynasore or left untreated were infected with S. pyogenes (MOI = 100) for the indicated periods or left uninfected. At the indicated time points total RNA was isolated, reverse-transcribed and analyzed by qPCR for IFN-β. HPRT was used for normalization. Values represent mean ± SD; n = 3. ( B ) BMDMs pretreated (for 45 min) with dynasore or left untreated were infected with CFSE-labeled (green) S. pyogenes (MOI = 100) and fixed at indicated time points. Extracellular S. pyogenes were stained with anti- S. pyogenes antibody (red). The percentages of internalized S. pyogenes were calculated based on the number of red and green fluorescing bacteria in a field. At least five fields were counted and SDs were calculated. ( C ) Fluorescence microscopy imaging of cells treated as described in (B). Total S. pyogenes labeled with CFSE (green) and extracellular S. pyogenes stained post-fixation with anti- S. pyogenes antibody (red), as well as merge are shown. Green cells in the merge image represent internalized bacteria. Representative panels are shown. ( D ) BMDMs pretreated (for 45 min) with bafilomycin A1 or left untreated were infected with S. pyogenes (MOI = 100). At indicated time points, supernatants were collected and IFN-β release was measured as described in (A). Values represent mean ± SD; n = 3. ( E ) BMDMs treated with dynasore, bafilomycin A or left untreated were infected with S. pyogenes (MOI = 100). At indicated time points, serial dilutions of cellular lysates were plated to count colony-forming units (CFUs) that are displayed as log CFU.

Techniques Used: Infection, Isolation, Real-time Polymerase Chain Reaction, Labeling, Staining, Fluorescence, Microscopy, Imaging

18) Product Images from "Endocytosis is required for Toll signaling and shaping of the Dorsal/NF-?B morphogen gradient during Drosophila embryogenesis"

Article Title: Endocytosis is required for Toll signaling and shaping of the Dorsal/NF-?B morphogen gradient during Drosophila embryogenesis

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

doi: 10.1073/pnas.1009157107

Inhibition of endocytosis ventrally reduces nuclear accumulation of Dorsal proximally and can alter the polarity of the Dorsal gradient. Live imaging of transgenic embryos expressing Dorsal-GFP at nuclear cycle 14. ( A ) Mock-injected control embryo expressing Dorsal-GFP showing the WT (wt) nuclear Dorsal gradient. ( B ) Embryo microinjected with Dynasore, a dynamin inhibitor, as a small bolus on the ventral midline in which nuclear accumulation of Dorsal is attenuated near the site of injection and slightly expanded dorsally (arrow). ( C ) Embryo injected with synthetic mRNA encoding Rab5S43N, a dominant negative form of Rab5 on the ventral midline. ( D ) Embryo injected as in C and allowed to develop for 40 min, illustrating a progressive shifting of nuclear translocation to the dorsal side of the embryo. ( E ) Rab5S43N ventrally injected embryo showing complete inversion of the Dorsal gradient. In all cases, ventral is down, dorsal is up, and red circles mark the site of injection of drug or synthetic mRNA.
Figure Legend Snippet: Inhibition of endocytosis ventrally reduces nuclear accumulation of Dorsal proximally and can alter the polarity of the Dorsal gradient. Live imaging of transgenic embryos expressing Dorsal-GFP at nuclear cycle 14. ( A ) Mock-injected control embryo expressing Dorsal-GFP showing the WT (wt) nuclear Dorsal gradient. ( B ) Embryo microinjected with Dynasore, a dynamin inhibitor, as a small bolus on the ventral midline in which nuclear accumulation of Dorsal is attenuated near the site of injection and slightly expanded dorsally (arrow). ( C ) Embryo injected with synthetic mRNA encoding Rab5S43N, a dominant negative form of Rab5 on the ventral midline. ( D ) Embryo injected as in C and allowed to develop for 40 min, illustrating a progressive shifting of nuclear translocation to the dorsal side of the embryo. ( E ) Rab5S43N ventrally injected embryo showing complete inversion of the Dorsal gradient. In all cases, ventral is down, dorsal is up, and red circles mark the site of injection of drug or synthetic mRNA.

Techniques Used: Inhibition, Imaging, Transgenic Assay, Expressing, Injection, Dominant Negative Mutation, Translocation Assay

19) 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

20) 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:

21) Product Images from "A Distinct Endocytic Mechanism of Functionalized-Silica Nanoparticles in Breast Cancer Stem Cells"

Article Title: A Distinct Endocytic Mechanism of Functionalized-Silica Nanoparticles in Breast Cancer Stem Cells

Journal: Scientific Reports

doi: 10.1038/s41598-017-16591-z

Percentage of cellular uptake of SiNPs-OH, SiNPs-NH 2 , and SiNPs-COOH in MCF-7 and BCSCs in the presence of different endocytic inhibitors. ( a ) and ( b ) Energy-dependent internalization of NPs; ( c ) cyto D; ( d ) CPZ; ( e ) nystatin; ( f ) nocodazole; ( g ) Dynasore; ( h ) Genistein; ( i ) poly I. The values are the mean ± SD from three independent experiments. *p
Figure Legend Snippet: Percentage of cellular uptake of SiNPs-OH, SiNPs-NH 2 , and SiNPs-COOH in MCF-7 and BCSCs in the presence of different endocytic inhibitors. ( a ) and ( b ) Energy-dependent internalization of NPs; ( c ) cyto D; ( d ) CPZ; ( e ) nystatin; ( f ) nocodazole; ( g ) Dynasore; ( h ) Genistein; ( i ) poly I. The values are the mean ± SD from three independent experiments. *p

Techniques Used:

22) 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

23) 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

24) Product Images from "How do megakaryocytic microparticles target and deliver cargo to alter the fate of hematopoietic stem cells?"

Article Title: How do megakaryocytic microparticles target and deliver cargo to alter the fate of hematopoietic stem cells?

Journal: Journal of controlled release : official journal of the Controlled Release Society

doi: 10.1016/j.jconrel.2016.12.021

Macropinocytosis, lipid-raft and dynamin are engaged in uptake of MkMP by HSPCs HSPCs were pre-incubated with specific inhibitors impacting MkMP uptake by HSPCs. The inhibitors used were methyl-β-cyclodextrin (MβCD), dimetylamiloride (DMA), chlorpromazine (CH), and dynasore, which target lipid raft, macropinocytosis, clathrin-dependent endocytosis and dynamin-dependent or independent endocytosis, respectively. To assess the impact of the inhibitors on MkMP uptake, we compared the outcomes from the inhibitor studies against the uptake of the following molecules (positive controls), cholera toxin subunit B (CT-B), dextran 10k (Dx), and transferrin (Tf), which are established to be taken up by lipid-raft mediated endocytosis, macropinocytosis, and clathrin-dependent endocytosis, respectively. Specifically, d3-HSPCs were pre-incubated with or without 5 mM MβCD, 10 µM DMA, 20 µM CH, or 80 µM dynasore for 45 min at 37 °C, before the coculture with (A–D) CFDA-SE-stained MkMP for 30 min, (B) CT-B, (C) Dx, or (D) Tf for 15 min at 37 °C. Uptake of MkMP, CT-B, Dx, or Tf were analyzed by flow cytometry. Data represent averages of 3 biological replicates ± standard error of mean. *, P
Figure Legend Snippet: Macropinocytosis, lipid-raft and dynamin are engaged in uptake of MkMP by HSPCs HSPCs were pre-incubated with specific inhibitors impacting MkMP uptake by HSPCs. The inhibitors used were methyl-β-cyclodextrin (MβCD), dimetylamiloride (DMA), chlorpromazine (CH), and dynasore, which target lipid raft, macropinocytosis, clathrin-dependent endocytosis and dynamin-dependent or independent endocytosis, respectively. To assess the impact of the inhibitors on MkMP uptake, we compared the outcomes from the inhibitor studies against the uptake of the following molecules (positive controls), cholera toxin subunit B (CT-B), dextran 10k (Dx), and transferrin (Tf), which are established to be taken up by lipid-raft mediated endocytosis, macropinocytosis, and clathrin-dependent endocytosis, respectively. Specifically, d3-HSPCs were pre-incubated with or without 5 mM MβCD, 10 µM DMA, 20 µM CH, or 80 µM dynasore for 45 min at 37 °C, before the coculture with (A–D) CFDA-SE-stained MkMP for 30 min, (B) CT-B, (C) Dx, or (D) Tf for 15 min at 37 °C. Uptake of MkMP, CT-B, Dx, or Tf were analyzed by flow cytometry. Data represent averages of 3 biological replicates ± standard error of mean. *, P

Techniques Used: Incubation, Staining, Flow Cytometry, Cytometry

25) 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

26) Product Images from "TLR4-dependent activation of dendritic cells by an HMGB1-derived peptide adjuvant"

Article Title: TLR4-dependent activation of dendritic cells by an HMGB1-derived peptide adjuvant

Journal: Journal of Translational Medicine

doi: 10.1186/1479-5876-12-211

TLR4, MyD88, and MyD88-dependent and -independent pathways are necessary for Hp91-mediated activation of antigen presenting cells. (A) BM-DCs from wild type (WT) or knockout mice were exposed to 200 μg/ml Hp91. Supernatants were analyzed for IL-6 by ELISA. Results are mean (±SEM) for N = 3-5. (B) J774 macrophages were stimulated for indicated times with 200 μg/ml Hp91, 10 ng/ml LPS or left untreated (M). Lysates were analyzed for p-p38 by WB. Blots were probed with anti-p-p38, anti-p38, and anti-GAPDH antibodies. One of N=4. (C) J774 macrophages were pretreated with media (Med), DMSO control (D), Dynasore (DYN) 80 μM, phenylarsine oxide (PAO) 2 μM or chlorpromazine (CP) 100 μM before incubation with 200 μg/ml Cp488-labeled Hp91 for 30 min. Cells were analyzed by flow cytometry. Results are mean (±SEM), N=4. (D) J774 macrophages were pretreated with DMSO control (D), SB203580 (SB) 20 μM, PD98059 (PD) 20 μM, TPCK 20 μM, Dynasore (DYN) 80 μM, phenylarsine oxide (PAO) 2 μM, or chlorpromazine (CP) 100 μM then exposed to Hp91. Supernatants were analyzed for IL-6 by ELISA. Data are mean (±SEM), N=3. (E) RAW 264.7 macrophages were pretreated with medium, DMSO control, or Dynasore (DYN) 80 μM prior to stimulation for indicated times with Hp91 (200 μg/ml), LPS (10 ng/ml) or media. Cell lysates were analyzed for p-IRF3. Immunoblots were probed with anti-p-IRF3, anti-IRF3, and anti-β-actin antibodies. (F) cDNA from stimulated J774 cells were evaluated for IFN-α2 mRNA by qPCR. Values are normalized against endogenous GAPDH controls, in duplicate. (G) Wildtype or MyD88-/- knockout mice were immunized with SIINFEKL peptide in PBS with or without Hp91. Splenocytes from immunized mice were cultured with SIINFEKL peptide (2.5 μg/ml). The number of IFN-γ-secreting cells was determined 18 h later. Data are mean (±SEM), 5–10 mice/group. *p
Figure Legend Snippet: TLR4, MyD88, and MyD88-dependent and -independent pathways are necessary for Hp91-mediated activation of antigen presenting cells. (A) BM-DCs from wild type (WT) or knockout mice were exposed to 200 μg/ml Hp91. Supernatants were analyzed for IL-6 by ELISA. Results are mean (±SEM) for N = 3-5. (B) J774 macrophages were stimulated for indicated times with 200 μg/ml Hp91, 10 ng/ml LPS or left untreated (M). Lysates were analyzed for p-p38 by WB. Blots were probed with anti-p-p38, anti-p38, and anti-GAPDH antibodies. One of N=4. (C) J774 macrophages were pretreated with media (Med), DMSO control (D), Dynasore (DYN) 80 μM, phenylarsine oxide (PAO) 2 μM or chlorpromazine (CP) 100 μM before incubation with 200 μg/ml Cp488-labeled Hp91 for 30 min. Cells were analyzed by flow cytometry. Results are mean (±SEM), N=4. (D) J774 macrophages were pretreated with DMSO control (D), SB203580 (SB) 20 μM, PD98059 (PD) 20 μM, TPCK 20 μM, Dynasore (DYN) 80 μM, phenylarsine oxide (PAO) 2 μM, or chlorpromazine (CP) 100 μM then exposed to Hp91. Supernatants were analyzed for IL-6 by ELISA. Data are mean (±SEM), N=3. (E) RAW 264.7 macrophages were pretreated with medium, DMSO control, or Dynasore (DYN) 80 μM prior to stimulation for indicated times with Hp91 (200 μg/ml), LPS (10 ng/ml) or media. Cell lysates were analyzed for p-IRF3. Immunoblots were probed with anti-p-IRF3, anti-IRF3, and anti-β-actin antibodies. (F) cDNA from stimulated J774 cells were evaluated for IFN-α2 mRNA by qPCR. Values are normalized against endogenous GAPDH controls, in duplicate. (G) Wildtype or MyD88-/- knockout mice were immunized with SIINFEKL peptide in PBS with or without Hp91. Splenocytes from immunized mice were cultured with SIINFEKL peptide (2.5 μg/ml). The number of IFN-γ-secreting cells was determined 18 h later. Data are mean (±SEM), 5–10 mice/group. *p

Techniques Used: Activation Assay, Knock-Out, Mouse Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Incubation, Labeling, Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction, Cell Culture

27) 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

28) Product Images from "ADP-Ribosylation Factor 6 Mediates E-Cadherin Recovery by Chemical Chaperones"

Article Title: ADP-Ribosylation Factor 6 Mediates E-Cadherin Recovery by Chemical Chaperones

Journal: PLoS ONE

doi: 10.1371/journal.pone.0023188

Endocytosis inhibition increases E-cadherin at the plasma membrane. CHO cells stably transduced with the empty vector (Mock) or with WT, R749W or E757K hEcadherin were treated with Dynasore or MiTMAB for 17h. (A) Clathrin, E-cadherin and Arf6 expression were analyzed in whole cell lysates by Western Blot. Actin was used as a loading control. The intensity of the bands was quantified and normalized against the untreated WT cells. The graphs show the average + SE of protein level, in three independent experiments. (B) After treatment, Transferrin 594 was added to cells. Nucleus was counterstained with DAPI. The pictures were taken under a 63× objective. (C) Cells were fixed and immunostained with anti-human E-cadherin antibody. Nucleus was counterstained with DAPI. The pictures were taken under a 40× objective. (D) Flow cytometry technique was used to assess E-cadherin cell surface expression. Each histogram represents the cell surface expression of E-cadherin in cells WT, R749W or E757K, treated with Dynasore (red) or MiTMAB (green) or untreated (blue). The black area in the histogram represents the cells that were not incubated with primary antibody, this sample was used as negative control. For each sample, the number of cells expressing surface E-cadherin was calculated. The mean fluorescence intensity was also quantified and normalized against the control of WT expressing cells. The graphs show the average + SE, n = 3 (* represents p≤0.05).
Figure Legend Snippet: Endocytosis inhibition increases E-cadherin at the plasma membrane. CHO cells stably transduced with the empty vector (Mock) or with WT, R749W or E757K hEcadherin were treated with Dynasore or MiTMAB for 17h. (A) Clathrin, E-cadherin and Arf6 expression were analyzed in whole cell lysates by Western Blot. Actin was used as a loading control. The intensity of the bands was quantified and normalized against the untreated WT cells. The graphs show the average + SE of protein level, in three independent experiments. (B) After treatment, Transferrin 594 was added to cells. Nucleus was counterstained with DAPI. The pictures were taken under a 63× objective. (C) Cells were fixed and immunostained with anti-human E-cadherin antibody. Nucleus was counterstained with DAPI. The pictures were taken under a 40× objective. (D) Flow cytometry technique was used to assess E-cadherin cell surface expression. Each histogram represents the cell surface expression of E-cadherin in cells WT, R749W or E757K, treated with Dynasore (red) or MiTMAB (green) or untreated (blue). The black area in the histogram represents the cells that were not incubated with primary antibody, this sample was used as negative control. For each sample, the number of cells expressing surface E-cadherin was calculated. The mean fluorescence intensity was also quantified and normalized against the control of WT expressing cells. The graphs show the average + SE, n = 3 (* represents p≤0.05).

Techniques Used: Inhibition, Stable Transfection, Transduction, Plasmid Preparation, Expressing, Western Blot, Flow Cytometry, Cytometry, Incubation, Negative Control, Fluorescence

29) Product Images from "A Positive Role of Cadherin in Wnt/?-Catenin Signalling during Epithelial-Mesenchymal Transition"

Article Title: A Positive Role of Cadherin in Wnt/?-Catenin Signalling during Epithelial-Mesenchymal Transition

Journal: PLoS ONE

doi: 10.1371/journal.pone.0023899

Stimulation of β-catenin signals by HGF is dependent on endocytosis. (A) Western blot analyses of the cytosolic fraction of MDCK cells after treatment with HGF and increasing amounts of Dynasore (50, 100, 200 and 400 µM) for 6 and 16 hours shows a dose-dependent reduction in cytosolic β-catenin accumulation by HGF. (B) Western blot analysis of cytosolic fraction of MDCK cells after treatment with HGF and Dynasore (100 and 400 µM) for 30 minutes. Blocking endocytosis by Dynasore does not interfere with HGF signalling-induced phsophorylation of Erk (p-Erk). (C–H) Confocal microscopy of MDCK cells showing E-cadherin (green) and either β-catenin (red in C,E,G) or transferrin receptor (red in D,F,H) after treatment with HGF without or with Dynasore (400 µM) for 16 hours. In the absence of HGF or Dynasore (C,D), β-catenin and E-cadherin are localised at the cell membrane (C, also shown in Fig. 2A,B ) while transferrin receptor is mostly in the subcortical area (D). HGF treatment (E,F) causes internalisation of β-catenin and E-cadherin (E, also shown in Fig. 2E,F ) and movement of transferrin receptor to a perinuclear location, where it colocalises with internalised E-cadherin (F). In the presence of Dynasore, the ability of HGF to induce internalisation of β-catenin and E-cadherin is abolished, and they both remain at the cell membrane (G). Scale bars; 20 µm. (I) TOPflash reporter assay in MDCK cells with control (c) or β-catenin (β-cat) transfection and HGF treatment (similar to Fig. 1F ), in the absence or presence of Dynasore (400 µM). There is a reduction in Wnt pathway readout by both HGF and HGF plus β-catenin in the presence of Dynasore. HGF and Dynasore were added to transfected cells for the final 16 hours of incubation. Data were all normalised to the control without Dynasore. (J–L) Confocal microscopy of MDCK cells transfected with Flag-tagged β-catenin (red), treated with HGF and Dynasore as indicated, counterstained with DAPI (blue). Exogenous β-catenin is localised at the cell membrane in the absence of HGF (J), whereas HGF-treated cells show exogenous β-catenin in the cytoplasm, both in the absence (K) and presence (L) of Dynasore. Scale bar; 20 µm. (M) TOPflash reporter assay in MDCK cells with β-catenin transfection and HGF and Dynasore (200 µM) treatment. Where applicable, HGF were first added to cells for 24 hours to make cells mesenchymal, before adding Dynasore for 16 hours (total 40 hours of HGF). Dynasore attenuates the effect of transfected β-catenin in the mesenchymally transformed cells. * P = 0.011. (N) Schematic diagram summarising the requirement for the release of an active form of β-catenin into the cytosol.
Figure Legend Snippet: Stimulation of β-catenin signals by HGF is dependent on endocytosis. (A) Western blot analyses of the cytosolic fraction of MDCK cells after treatment with HGF and increasing amounts of Dynasore (50, 100, 200 and 400 µM) for 6 and 16 hours shows a dose-dependent reduction in cytosolic β-catenin accumulation by HGF. (B) Western blot analysis of cytosolic fraction of MDCK cells after treatment with HGF and Dynasore (100 and 400 µM) for 30 minutes. Blocking endocytosis by Dynasore does not interfere with HGF signalling-induced phsophorylation of Erk (p-Erk). (C–H) Confocal microscopy of MDCK cells showing E-cadherin (green) and either β-catenin (red in C,E,G) or transferrin receptor (red in D,F,H) after treatment with HGF without or with Dynasore (400 µM) for 16 hours. In the absence of HGF or Dynasore (C,D), β-catenin and E-cadherin are localised at the cell membrane (C, also shown in Fig. 2A,B ) while transferrin receptor is mostly in the subcortical area (D). HGF treatment (E,F) causes internalisation of β-catenin and E-cadherin (E, also shown in Fig. 2E,F ) and movement of transferrin receptor to a perinuclear location, where it colocalises with internalised E-cadherin (F). In the presence of Dynasore, the ability of HGF to induce internalisation of β-catenin and E-cadherin is abolished, and they both remain at the cell membrane (G). Scale bars; 20 µm. (I) TOPflash reporter assay in MDCK cells with control (c) or β-catenin (β-cat) transfection and HGF treatment (similar to Fig. 1F ), in the absence or presence of Dynasore (400 µM). There is a reduction in Wnt pathway readout by both HGF and HGF plus β-catenin in the presence of Dynasore. HGF and Dynasore were added to transfected cells for the final 16 hours of incubation. Data were all normalised to the control without Dynasore. (J–L) Confocal microscopy of MDCK cells transfected with Flag-tagged β-catenin (red), treated with HGF and Dynasore as indicated, counterstained with DAPI (blue). Exogenous β-catenin is localised at the cell membrane in the absence of HGF (J), whereas HGF-treated cells show exogenous β-catenin in the cytoplasm, both in the absence (K) and presence (L) of Dynasore. Scale bar; 20 µm. (M) TOPflash reporter assay in MDCK cells with β-catenin transfection and HGF and Dynasore (200 µM) treatment. Where applicable, HGF were first added to cells for 24 hours to make cells mesenchymal, before adding Dynasore for 16 hours (total 40 hours of HGF). Dynasore attenuates the effect of transfected β-catenin in the mesenchymally transformed cells. * P = 0.011. (N) Schematic diagram summarising the requirement for the release of an active form of β-catenin into the cytosol.

Techniques Used: Western Blot, Blocking Assay, Confocal Microscopy, Reporter Assay, Transfection, Incubation, Transformation Assay

30) Product Images from "Amino Acid Differences in the 1753-to-1851 Region of TcdB Influence Variations in TcdB1 and TcdB2 Cell Entry"

Article Title: Amino Acid Differences in the 1753-to-1851 Region of TcdB Influence Variations in TcdB1 and TcdB2 Cell Entry

Journal: mSphere

doi: 10.1128/mSphere.00268-17

TcdB2 B2′B3 localizes to acidified compartments. Transmitted fluorescence and maximum-intensity projections of pHrodo-labeled TcdB fragments (green) (used at 250 nM) incubated with CHO-K1 cells for 1 h at 37°C. DNA was stained with 0.5 µg/ml Hoechst 33258 (blue). (A to C) TcdB1 B2′B3 incubated with the vehicle control. (D to F) TcdB2 B2′B3 incubated with the vehicle control. (G to I) TcdB2 B2′B3 incubated with 80 μM dynasore. (J to L) TcdB2 B3 incubated with the vehicle control. Panels A, D, G, and J show transmitted fluorescence. Panels C, F, I, and L are ×2 magnifications of the inserts in panels B, E, H, and K, respectively.
Figure Legend Snippet: TcdB2 B2′B3 localizes to acidified compartments. Transmitted fluorescence and maximum-intensity projections of pHrodo-labeled TcdB fragments (green) (used at 250 nM) incubated with CHO-K1 cells for 1 h at 37°C. DNA was stained with 0.5 µg/ml Hoechst 33258 (blue). (A to C) TcdB1 B2′B3 incubated with the vehicle control. (D to F) TcdB2 B2′B3 incubated with the vehicle control. (G to I) TcdB2 B2′B3 incubated with 80 μM dynasore. (J to L) TcdB2 B3 incubated with the vehicle control. Panels A, D, G, and J show transmitted fluorescence. Panels C, F, I, and L are ×2 magnifications of the inserts in panels B, E, H, and K, respectively.

Techniques Used: Fluorescence, Labeling, Incubation, Staining

Effective TcdB cell interactions correlate with endocytosis. (A, B) Representative confocal maximum-intensity projections of CHO-K1 cells stained with 250 nM Alexa Fluor 647-labeled TcdB1 B2′B3 (red) (A) or Alexa Fluor 647-labeled TcdB2 B2′B3 (red) (B). Ten micromolar calcein AM (green) was used to counterstain the cytoplasm of live cells with intact membranes, and 0.5 μg/ml Hoechst 33258 (blue) was used to visualize the nucleus. (C) Quantification of the images in panels A and B, expressed as corrected total cell fluorescence. (D) Representative immunoblotting of full-length TcdB1 and TcdB2 associated with CHO-K1 cells in the presence or absence the dynamin inhibitor dynasore (80 μM). Incubations were carried out for 30 min at 37°C. (E) Quantification of data presented in panel D, expressed as relative band density. (F) Representative immunoblotting of full-length TcdB1 and TcdB2 associated with CHO-K1 cells when incubations were carried out for 30 min at a temperature that permits (37°C) or inhibits (ice) endocytosis. (G) Quantification of data presented in panel F, expressed as relative band density. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Figure Legend Snippet: Effective TcdB cell interactions correlate with endocytosis. (A, B) Representative confocal maximum-intensity projections of CHO-K1 cells stained with 250 nM Alexa Fluor 647-labeled TcdB1 B2′B3 (red) (A) or Alexa Fluor 647-labeled TcdB2 B2′B3 (red) (B). Ten micromolar calcein AM (green) was used to counterstain the cytoplasm of live cells with intact membranes, and 0.5 μg/ml Hoechst 33258 (blue) was used to visualize the nucleus. (C) Quantification of the images in panels A and B, expressed as corrected total cell fluorescence. (D) Representative immunoblotting of full-length TcdB1 and TcdB2 associated with CHO-K1 cells in the presence or absence the dynamin inhibitor dynasore (80 μM). Incubations were carried out for 30 min at 37°C. (E) Quantification of data presented in panel D, expressed as relative band density. (F) Representative immunoblotting of full-length TcdB1 and TcdB2 associated with CHO-K1 cells when incubations were carried out for 30 min at a temperature that permits (37°C) or inhibits (ice) endocytosis. (G) Quantification of data presented in panel F, expressed as relative band density. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Techniques Used: Staining, Labeling, Fluorescence

31) 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

32) Product Images from "Deciphering the Contribution of Human Meningothelial Cells to the Inflammatory and Antimicrobial Response at the Meninges"

Article Title: Deciphering the Contribution of Human Meningothelial Cells to the Inflammatory and Antimicrobial Response at the Meninges

Journal: Infection and Immunity

doi: 10.1128/IAI.00477-13

Role of endocytosis in TLR4 signaling. Meningothelial cells were treated with cytochalasin D (CytoD), latrunculin A (LTCa), or Dynasore or were given no treatment for 1 h before stimulation with MC58 or LPS. IL-6, IL-8, CXCL10, and CCL5 secretion was
Figure Legend Snippet: Role of endocytosis in TLR4 signaling. Meningothelial cells were treated with cytochalasin D (CytoD), latrunculin A (LTCa), or Dynasore or were given no treatment for 1 h before stimulation with MC58 or LPS. IL-6, IL-8, CXCL10, and CCL5 secretion was

Techniques Used:

33) Product Images from "NOX2 oxidase expressed in endosomes promotes cell proliferation and prostate tumour development"

Article Title: NOX2 oxidase expressed in endosomes promotes cell proliferation and prostate tumour development

Journal: Oncotarget

doi: 10.18632/oncotarget.26237

VEGF significantly increases endothelial cell proliferation in a H 2 O 2 - dependent manner and is further dependent on endocytosis, endosomal acidification and NOX2 activity ( A – F ) The proportion of HMEC-1 per well after 24 hr treatment with either PBS or VEGF-A (30 ng/mL) in the absence or presence of either (A) Dynasore (Dyna; 100 µM), (B) pitstop 2 (Pit; 30 µM), (C) bafilomycin A (Baf; 10 nM), (D) catalase (Cat; 1000 U/mL), (E) SOD (100 U/mL) or (F) apocynin (Apo; 300 µM). ( G ) Graph shows the effects of VEGF-A (10 and 30 ng/mL) on WT and NOX2 -/- mouse lung endothelial cell proliferation after 24 hr expressed as percentages of the PBS control. Data are mean ± SEM for n = 5-7 experiments. * P
Figure Legend Snippet: VEGF significantly increases endothelial cell proliferation in a H 2 O 2 - dependent manner and is further dependent on endocytosis, endosomal acidification and NOX2 activity ( A – F ) The proportion of HMEC-1 per well after 24 hr treatment with either PBS or VEGF-A (30 ng/mL) in the absence or presence of either (A) Dynasore (Dyna; 100 µM), (B) pitstop 2 (Pit; 30 µM), (C) bafilomycin A (Baf; 10 nM), (D) catalase (Cat; 1000 U/mL), (E) SOD (100 U/mL) or (F) apocynin (Apo; 300 µM). ( G ) Graph shows the effects of VEGF-A (10 and 30 ng/mL) on WT and NOX2 -/- mouse lung endothelial cell proliferation after 24 hr expressed as percentages of the PBS control. Data are mean ± SEM for n = 5-7 experiments. * P

Techniques Used: Activity Assay

Co-location of early endosomes and VEGFR2 in the presence of VEGF is endocytosis-dependent ( A ) Confocal immunofluorescent images showing EEA1 and VEGFR2 co-location within HMEC-1 in and around the DAPI-stained nucleus after 30 min incubation with either PBS, VEGF-A (30 ng/mL), VEGF-A (30 ng/mL) plus dynasore (Dyna; 100 µM) or VEGF-A (30 ng/mL) plus Pitstop 2 (Pit; 30 µM). ( B ) Graphs show the degree of co-location within these cells in both groups expressed as percentage of total EEA1 positive staining. Data is representative of > 50 cells imaged across 4 experiments and are shown as mean ± SEM. * P
Figure Legend Snippet: Co-location of early endosomes and VEGFR2 in the presence of VEGF is endocytosis-dependent ( A ) Confocal immunofluorescent images showing EEA1 and VEGFR2 co-location within HMEC-1 in and around the DAPI-stained nucleus after 30 min incubation with either PBS, VEGF-A (30 ng/mL), VEGF-A (30 ng/mL) plus dynasore (Dyna; 100 µM) or VEGF-A (30 ng/mL) plus Pitstop 2 (Pit; 30 µM). ( B ) Graphs show the degree of co-location within these cells in both groups expressed as percentage of total EEA1 positive staining. Data is representative of > 50 cells imaged across 4 experiments and are shown as mean ± SEM. * P

Techniques Used: Staining, Incubation

34) Product Images from "Inhibition of CBL-B protects from lethal C. albicans sepsis"

Article Title: Inhibition of CBL-B protects from lethal C. albicans sepsis

Journal: Nature medicine

doi: 10.1038/nm.4134

Cblb controls anti-fungal activities of dendritic cells and macrophages. ( a ) Killing capacity of BM-neutrophils, BM-M, BM-DC, BM-monocytes, or splenic DC as assessed by co-culture with C. albicans . Assays performed in quadruplicates. ( b,c ) Killing capacity of BM-DC in the presence of (b) the phagocytosis inhibitor Dynasore or an anti-Dectin-1 antibody, or ( c ) the SYK inhibitor R406. ( d,e ) ROS production by intraperitoneal immune infiltrates of Cblb +/+ or Cblb Δ/Δ mice 24 h after intraperitoneal C. albicans infection (5 * 10 6 CFU/21.5 g body weight). Plots depict ( d ) ROS production by immune infiltrates without re-stimulation or ( e ) with re-stimulation with C. albicans . Experiments performed in triplicates. ( f ) Killing capacity of immune infiltrates from ( d,e ). ( g,h ) ROS production of ( g ) monocytes/macrophages or ( h ) neutrophils FACS sorted from infiltrates in ( d,e ). ( i,j ) Cblb +/+ or Cblb Δ/Δ mice were injected with PBS liposomes or clodronate liposomes 24 h before and 24 h after intravenous infection with C. albicans (10 5 CFU/21.5 g body weight) and monitored for ( i ) weight loss, as compared to starting weight ( P values assessed by two-way ANOVA), or ( j ) survival ( P values assessed with log rank test). n = 5 for PBS liposome treated, n = 8 for clodronate liposome treated cohorts. For panels ( a-c,f,i ) data are shown as means ± standard deviation. For panels ( a,b ) 1 representative of 5, for panels ( c-j ) 1 representative of 3 independent experiments is shown. * P
Figure Legend Snippet: Cblb controls anti-fungal activities of dendritic cells and macrophages. ( a ) Killing capacity of BM-neutrophils, BM-M, BM-DC, BM-monocytes, or splenic DC as assessed by co-culture with C. albicans . Assays performed in quadruplicates. ( b,c ) Killing capacity of BM-DC in the presence of (b) the phagocytosis inhibitor Dynasore or an anti-Dectin-1 antibody, or ( c ) the SYK inhibitor R406. ( d,e ) ROS production by intraperitoneal immune infiltrates of Cblb +/+ or Cblb Δ/Δ mice 24 h after intraperitoneal C. albicans infection (5 * 10 6 CFU/21.5 g body weight). Plots depict ( d ) ROS production by immune infiltrates without re-stimulation or ( e ) with re-stimulation with C. albicans . Experiments performed in triplicates. ( f ) Killing capacity of immune infiltrates from ( d,e ). ( g,h ) ROS production of ( g ) monocytes/macrophages or ( h ) neutrophils FACS sorted from infiltrates in ( d,e ). ( i,j ) Cblb +/+ or Cblb Δ/Δ mice were injected with PBS liposomes or clodronate liposomes 24 h before and 24 h after intravenous infection with C. albicans (10 5 CFU/21.5 g body weight) and monitored for ( i ) weight loss, as compared to starting weight ( P values assessed by two-way ANOVA), or ( j ) survival ( P values assessed with log rank test). n = 5 for PBS liposome treated, n = 8 for clodronate liposome treated cohorts. For panels ( a-c,f,i ) data are shown as means ± standard deviation. For panels ( a,b ) 1 representative of 5, for panels ( c-j ) 1 representative of 3 independent experiments is shown. * P

Techniques Used: Co-Culture Assay, Mouse Assay, Infection, FACS, Injection, Standard Deviation

35) 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

36) Product Images from "Reduction in dynamin-2 is implicated in ischaemic cardiac arrhythmias"

Article Title: Reduction in dynamin-2 is implicated in ischaemic cardiac arrhythmias

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/jcmm.12335

DNM2 deficiency changes AP in ventricular myocytes. ( A ) Representative AP recordings from Ctrl (control vector) and DNM2-K44A overexpression cells. ( B ) Representative AP recordings from DMSO-and dynasore-treated myocytes. ( C – F ) Statistic analysis for action potential amplitude (APA) action potential duration at 20% (APD 20 ), APD 50 , APD 90 . * P
Figure Legend Snippet: DNM2 deficiency changes AP in ventricular myocytes. ( A ) Representative AP recordings from Ctrl (control vector) and DNM2-K44A overexpression cells. ( B ) Representative AP recordings from DMSO-and dynasore-treated myocytes. ( C – F ) Statistic analysis for action potential amplitude (APA) action potential duration at 20% (APD 20 ), APD 50 , APD 90 . * P

Techniques Used: Plasmid Preparation, Over Expression

37) Product Images from "Keratins control intercellular adhesion involving PKC-?-mediated desmoplakin phosphorylation"

Article Title: Keratins control intercellular adhesion involving PKC-?-mediated desmoplakin phosphorylation

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201208162

Keratins affect dynamin-dependent endocytosis. (A–D′) DP staining revealed almost complete desmosome internalization in KtyII −/− cells after EGTA treatment (B′), whereas internalization is slower in controls (A′). Blocking endocytosis or reexpression of K5/14 in KtyII −/− cells induced relocalization of junctional proteins to PM in high Ca 2+ conditions (C and D) as well as 1 h after EGTA incubation (C′ and D′). (E) Ratios of average pixel intensities for DP at PM and cytosol ( n = 50). (F) WB of total protein lysates after EGTA treatment demonstrated accelerated Dsg2 degradation in KtyII −/− cells, reverted by reexpression of K5/14. Data (mean ± SEM, n = 3) are fold change relative to control. (G) Surface biotinylation and WB showed increased surface localization of Dsg2 after Dynasore treatment in KtyII −/− cells. The first two control lanes (WT and KtyII −/− with no drug treatment) are the same as shown in Fig. 3 H because the treated samples were loaded on the same blot. (H–H′′) Blocking endocytosis in KtyII −/− cells restored mechanical strength after dispase treatment ( n = 3). (I) IP with pSer antibodies followed by WB: reduction of P-DP in KtyII −/− cells after Dynasore treatment ( n = 2). (J) Model suggesting keratins regulate PKC-α–mediated phosphorylation of DP via RACK1.
Figure Legend Snippet: Keratins affect dynamin-dependent endocytosis. (A–D′) DP staining revealed almost complete desmosome internalization in KtyII −/− cells after EGTA treatment (B′), whereas internalization is slower in controls (A′). Blocking endocytosis or reexpression of K5/14 in KtyII −/− cells induced relocalization of junctional proteins to PM in high Ca 2+ conditions (C and D) as well as 1 h after EGTA incubation (C′ and D′). (E) Ratios of average pixel intensities for DP at PM and cytosol ( n = 50). (F) WB of total protein lysates after EGTA treatment demonstrated accelerated Dsg2 degradation in KtyII −/− cells, reverted by reexpression of K5/14. Data (mean ± SEM, n = 3) are fold change relative to control. (G) Surface biotinylation and WB showed increased surface localization of Dsg2 after Dynasore treatment in KtyII −/− cells. The first two control lanes (WT and KtyII −/− with no drug treatment) are the same as shown in Fig. 3 H because the treated samples were loaded on the same blot. (H–H′′) Blocking endocytosis in KtyII −/− cells restored mechanical strength after dispase treatment ( n = 3). (I) IP with pSer antibodies followed by WB: reduction of P-DP in KtyII −/− cells after Dynasore treatment ( n = 2). (J) Model suggesting keratins regulate PKC-α–mediated phosphorylation of DP via RACK1.

Techniques Used: Staining, Blocking Assay, Incubation, Western Blot

38) Product Images from "Pro-inflammatory hepatic macrophages generate ROS through NADPH oxidase 2 via endocytosis of monomeric TLR4–MD2 complex"

Article Title: Pro-inflammatory hepatic macrophages generate ROS through NADPH oxidase 2 via endocytosis of monomeric TLR4–MD2 complex

Journal: Nature Communications

doi: 10.1038/s41467-017-02325-2

Palmitate treatment increases ROS generation in human CD68 low CD14 high monocytes via endocytosis of palmitate/TLR4 complex. a Healthy human peripheral blood mononuclear cells (PBMCs) and isolated liver mononuclear cells (MNCs) from non-tumor lesions of HCC (HBV origin) were subjected to flow cytometry and assessment of ROS generation, respectively. b After the reaction between each protein and BODIPY-labeled fluorescent fatty acid analog (C16-BODIPY) under the indicated conditions, the samples loaded at Native gradient PAGE (4–15%) were visualized with illumination (488 nm) and Coomassie staining. c Healthy human PBMCs were treated with palmitate ± dynasore. Then, these cells were subjected to assessment of ROS generation and qRT–PCR analyses. d Freshly isolated liver MNCs from non-tumor liver lesions of primary HCC and biopsy lesions of fatty liver were subjected to flow cytometry and qRT–PCR analyses. Data are representative of three independent experiments using PBMC of healthy controls ( n = 5) and liver MNCs of HBV ( n = 3), cryptogenic HCC ( n = 1) and fatty liver ( n = 1) patients a , c , d . Data are expressed as the mean ± s.e.m. and analyzed by Student’s t -test or one-way analysis of variance, * P
Figure Legend Snippet: Palmitate treatment increases ROS generation in human CD68 low CD14 high monocytes via endocytosis of palmitate/TLR4 complex. a Healthy human peripheral blood mononuclear cells (PBMCs) and isolated liver mononuclear cells (MNCs) from non-tumor lesions of HCC (HBV origin) were subjected to flow cytometry and assessment of ROS generation, respectively. b After the reaction between each protein and BODIPY-labeled fluorescent fatty acid analog (C16-BODIPY) under the indicated conditions, the samples loaded at Native gradient PAGE (4–15%) were visualized with illumination (488 nm) and Coomassie staining. c Healthy human PBMCs were treated with palmitate ± dynasore. Then, these cells were subjected to assessment of ROS generation and qRT–PCR analyses. d Freshly isolated liver MNCs from non-tumor liver lesions of primary HCC and biopsy lesions of fatty liver were subjected to flow cytometry and qRT–PCR analyses. Data are representative of three independent experiments using PBMC of healthy controls ( n = 5) and liver MNCs of HBV ( n = 3), cryptogenic HCC ( n = 1) and fatty liver ( n = 1) patients a , c , d . Data are expressed as the mean ± s.e.m. and analyzed by Student’s t -test or one-way analysis of variance, * P

Techniques Used: Isolation, Flow Cytometry, Cytometry, Labeling, Polyacrylamide Gel Electrophoresis, Staining, Quantitative RT-PCR

Palmitate treatment increases ROS generation in CD11b + F4/80 low macrophages in TLR4 and NOX2-dependent manners. a Palmitate-mediated generation of reactive oxygen species (ROS) was monitored by DCF fluorescence in freshly isolated CD11b + F4/80 high Kupffer cells and CD11b + F4/80 low macrophages of WT , Nox2 knockout (KO) and Tlr4 KO mice. b Freshly isolated macrophages and Kupffer cells of WT mice were subjected to quantitative real-time PCR (qRT–PCR) analyses. c Isolated macrophages of WT mice were treated with palmitate with or without dynasore (Dyn) treatment. d RAW 264.7 macrophages were subjected to flow cytometry analyses. e , f RAW 264.7 macrophages were treated with palmitate (200 μM) ± dynasore (80 μM) for 1 h. Then, these cells were subjected to ROS generation assays, qRT–PCR analyses and immunostaining with antibodies of TLR4 and NOX2. Bar = 10 μm. Dotted white line indicates cell boundaries. Solid white rectangles are magnified. Data are representative of three independent experiments in vitro using isolated liver immune cells from 3 ( a – c ) mice per group. Data are expressed as the mean ± s.e.m. and analyzed by Student’s t -test or one-way analysis of variance, * P
Figure Legend Snippet: Palmitate treatment increases ROS generation in CD11b + F4/80 low macrophages in TLR4 and NOX2-dependent manners. a Palmitate-mediated generation of reactive oxygen species (ROS) was monitored by DCF fluorescence in freshly isolated CD11b + F4/80 high Kupffer cells and CD11b + F4/80 low macrophages of WT , Nox2 knockout (KO) and Tlr4 KO mice. b Freshly isolated macrophages and Kupffer cells of WT mice were subjected to quantitative real-time PCR (qRT–PCR) analyses. c Isolated macrophages of WT mice were treated with palmitate with or without dynasore (Dyn) treatment. d RAW 264.7 macrophages were subjected to flow cytometry analyses. e , f RAW 264.7 macrophages were treated with palmitate (200 μM) ± dynasore (80 μM) for 1 h. Then, these cells were subjected to ROS generation assays, qRT–PCR analyses and immunostaining with antibodies of TLR4 and NOX2. Bar = 10 μm. Dotted white line indicates cell boundaries. Solid white rectangles are magnified. Data are representative of three independent experiments in vitro using isolated liver immune cells from 3 ( a – c ) mice per group. Data are expressed as the mean ± s.e.m. and analyzed by Student’s t -test or one-way analysis of variance, * P

Techniques Used: Fluorescence, Isolation, Knock-Out, Mouse Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Flow Cytometry, Cytometry, Immunostaining, In Vitro

39) Product Images from "Infection of XC Cells by MLVs and Ebola Virus Is Endosome-Dependent but Acidification-Independent"

Article Title: Infection of XC Cells by MLVs and Ebola Virus Is Endosome-Dependent but Acidification-Independent

Journal: PLoS ONE

doi: 10.1371/journal.pone.0026180

Binding of the MLV vectors to target cells is not affected by ConA, CA-074Me, or dynasore treatments. (A) Binding of the Eco-MLV vector particles to TE671 (upper panel) and TE671/mCAT1 (lower panel) was analyzed. (B) XC cells were pretreated with DMSO, ConA, CA-074Me, or dynasore, and were incubated with the Eco-, Ampho-, Poly-, or Xeno-MLV vector. The fluorescence intensities obtained with DMSO-treated cells were set to 100%. These experiments were repeated in triplicate, and values are shown as the mean +/− SD.
Figure Legend Snippet: Binding of the MLV vectors to target cells is not affected by ConA, CA-074Me, or dynasore treatments. (A) Binding of the Eco-MLV vector particles to TE671 (upper panel) and TE671/mCAT1 (lower panel) was analyzed. (B) XC cells were pretreated with DMSO, ConA, CA-074Me, or dynasore, and were incubated with the Eco-, Ampho-, Poly-, or Xeno-MLV vector. The fluorescence intensities obtained with DMSO-treated cells were set to 100%. These experiments were repeated in triplicate, and values are shown as the mean +/− SD.

Techniques Used: Binding Assay, Plasmid Preparation, Incubation, Fluorescence

Ebola virus infection in XC cells is pH-independent. TE671 and XC cells were pretreated with ConA (panel A), CA-074Me (panel B), or dynasore (panel C), and were inoculated with the Ebola virus GP- or VSV-G-pseudotyped MLV vector. The transduction units obtained with DMSO-treated cells were set to 100%. These experiments were repeated in triplicate, and results are shown as the mean +/− SD. Asterisks indicate statistically significant differences compared to the value from the DMSO-treated cells.
Figure Legend Snippet: Ebola virus infection in XC cells is pH-independent. TE671 and XC cells were pretreated with ConA (panel A), CA-074Me (panel B), or dynasore (panel C), and were inoculated with the Ebola virus GP- or VSV-G-pseudotyped MLV vector. The transduction units obtained with DMSO-treated cells were set to 100%. These experiments were repeated in triplicate, and results are shown as the mean +/− SD. Asterisks indicate statistically significant differences compared to the value from the DMSO-treated cells.

Techniques Used: Infection, Plasmid Preparation, Transduction

MLV infections are inhibited by dynasore. (A) XC and NIH3T3 cells were pretreated with dynasore and were inoculated with the Eco-MLV vector diluted with fresh, TE671 cell conditioned, or cathepsin B (100 ng/ml)-containing medium. (B) XC and NIH3T3 cells were treated with dynasore, washed, and cultured for 48 h. Cells resistant to trypan blue staining were quantified. (C) TE671, TE671/mCAT1, or TE671/CD4 cells were pretreated with dynasore and were inoculated with the Eco-, Ampho-, Poly-, Xeno-, XMRV-, VSV-MLV, or HXB2-HIV-1 vector. (D) XC cells were pretreated with dynasore and were inoculated with the Ampho-, Poly-, Xeno-, XMRV-, or VSV-MLV vector. The transduction units obtained with DMSO-treated cells were set to 100%. These experiments were repeated in triplicate, and results are shown as the mean +/− SD. Asterisks indicate statistically significant differences compared to the value from the DMSO-treated cells.
Figure Legend Snippet: MLV infections are inhibited by dynasore. (A) XC and NIH3T3 cells were pretreated with dynasore and were inoculated with the Eco-MLV vector diluted with fresh, TE671 cell conditioned, or cathepsin B (100 ng/ml)-containing medium. (B) XC and NIH3T3 cells were treated with dynasore, washed, and cultured for 48 h. Cells resistant to trypan blue staining were quantified. (C) TE671, TE671/mCAT1, or TE671/CD4 cells were pretreated with dynasore and were inoculated with the Eco-, Ampho-, Poly-, Xeno-, XMRV-, VSV-MLV, or HXB2-HIV-1 vector. (D) XC cells were pretreated with dynasore and were inoculated with the Ampho-, Poly-, Xeno-, XMRV-, or VSV-MLV vector. The transduction units obtained with DMSO-treated cells were set to 100%. These experiments were repeated in triplicate, and results are shown as the mean +/− SD. Asterisks indicate statistically significant differences compared to the value from the DMSO-treated cells.

Techniques Used: Plasmid Preparation, Cell Culture, Staining, Transduction

40) Product Images from "APPL endosomes are not obligatory endocytic intermediates but act as stable cargo-sorting compartments"

Article Title: APPL endosomes are not obligatory endocytic intermediates but act as stable cargo-sorting compartments

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201311117

Cargo internalization into APPL endosomes is clathrin dependent but their biogenesis is not. (A) Silencing of CHC by RNAi in HeLa cells assessed by Western blot in comparison to EEA1, APPL1, and Rab5 as controls. (B) Internalization of biotinylated Tf (b-Tf) (after 30 min of continuous uptake) is inhibited upon CHC knockdown. The amounts of b-Tf in cell lysates were quantified by electrochemiluminescence. (C ) Knockdown of CHC decreased colocalization of Tf to EEA1 (red) and APPL1 (blue). Colocalization was quantified after 3.5-min chase after 0.5-min internalization pulse of Tf. (D–F) Knockdown of clathrin inhibits Tf uptake but does not affect the number of APPL1-positive vesicles. Example images of endogenous APPL1 and fluorescent Tf at 3.5-min chase after the 30-s internalization pulse in control and clathrin-depleted cells (D). Inset presents full image, yellow rectangle depicts zoomed part. The numbers of vesicles marked by APPL1 (red), Tf (blue) or EGF (green) (E) and their integral intensities (F) are plotted (quantifications based on 80 images and ∼320,000 APPL1 endosomes). (G–I) Dynasore treatment (from 10 to 60 min) does not affect the number of APPL1-positive vesicles but progressively suppresses Tf uptake (10 min of Tf internalization). (G) Example images of HeLa cells treated with Dynasore (80 µM) for 60 min. The numbers of vesicles marked by APPL1 (red) and Tf (blue) (H) and their integral intensities (I) in cells pretreated with Dynasore for the indicated times are plotted (quantifications based on 10 images, ∼110 cells, and ∼45,000 APPL1 endosomes). Bars: (D and G, inset) 10 µm.
Figure Legend Snippet: Cargo internalization into APPL endosomes is clathrin dependent but their biogenesis is not. (A) Silencing of CHC by RNAi in HeLa cells assessed by Western blot in comparison to EEA1, APPL1, and Rab5 as controls. (B) Internalization of biotinylated Tf (b-Tf) (after 30 min of continuous uptake) is inhibited upon CHC knockdown. The amounts of b-Tf in cell lysates were quantified by electrochemiluminescence. (C ) Knockdown of CHC decreased colocalization of Tf to EEA1 (red) and APPL1 (blue). Colocalization was quantified after 3.5-min chase after 0.5-min internalization pulse of Tf. (D–F) Knockdown of clathrin inhibits Tf uptake but does not affect the number of APPL1-positive vesicles. Example images of endogenous APPL1 and fluorescent Tf at 3.5-min chase after the 30-s internalization pulse in control and clathrin-depleted cells (D). Inset presents full image, yellow rectangle depicts zoomed part. The numbers of vesicles marked by APPL1 (red), Tf (blue) or EGF (green) (E) and their integral intensities (F) are plotted (quantifications based on 80 images and ∼320,000 APPL1 endosomes). (G–I) Dynasore treatment (from 10 to 60 min) does not affect the number of APPL1-positive vesicles but progressively suppresses Tf uptake (10 min of Tf internalization). (G) Example images of HeLa cells treated with Dynasore (80 µM) for 60 min. The numbers of vesicles marked by APPL1 (red) and Tf (blue) (H) and their integral intensities (I) in cells pretreated with Dynasore for the indicated times are plotted (quantifications based on 10 images, ∼110 cells, and ∼45,000 APPL1 endosomes). Bars: (D and G, inset) 10 µm.

Techniques Used: Western Blot, Electrochemiluminescence

41) Product Images from "The VEGF rise in blood of bevacizumab patients is not based on tumor escape but a host-blockade of VEGF clearance"

Article Title: The VEGF rise in blood of bevacizumab patients is not based on tumor escape but a host-blockade of VEGF clearance

Journal: Oncotarget

doi: 10.18632/oncotarget.11084

Loss of VEGF from endothelial culture supernatant A. 100 pg/ml of hrVEGF-165 were incubated with unconditioned (control), pre-conditioned (supernatant) EC medium or were directly added to confluent EC cultures (cells). The VEGF concentration was determined in the medium by ELISA after 1, 4 and 24 h of incubation. B. Furthermore, loss of VEGF was determined for subconfluent (proliferating) EC cultures. The possible effect of protein adherence to plastic was assessed by comparing VEGF concentrations in unconditioned medium without (control) or with (plastic adherence) incubation in an empty culture well for 24 h. C. VEGF loss from culture supernatant is due to internalization by ECs. When Pitstop 2 (33 μM) or Dynasore (250 μM) were added to EC cultures supplied with 100 pg/ml hrVEGF, the loss of VEGF from supernatant (after 4 h of incubation) was prevented. (* p
Figure Legend Snippet: Loss of VEGF from endothelial culture supernatant A. 100 pg/ml of hrVEGF-165 were incubated with unconditioned (control), pre-conditioned (supernatant) EC medium or were directly added to confluent EC cultures (cells). The VEGF concentration was determined in the medium by ELISA after 1, 4 and 24 h of incubation. B. Furthermore, loss of VEGF was determined for subconfluent (proliferating) EC cultures. The possible effect of protein adherence to plastic was assessed by comparing VEGF concentrations in unconditioned medium without (control) or with (plastic adherence) incubation in an empty culture well for 24 h. C. VEGF loss from culture supernatant is due to internalization by ECs. When Pitstop 2 (33 μM) or Dynasore (250 μM) were added to EC cultures supplied with 100 pg/ml hrVEGF, the loss of VEGF from supernatant (after 4 h of incubation) was prevented. (* p

Techniques Used: Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay

42) Product Images from "Flotillin-mediated endocytic events dictate cell-type specific responses to Semaphorin 3A"

Article Title: Flotillin-mediated endocytic events dictate cell-type specific responses to Semaphorin 3A

Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

doi: 10.1523/JNEUROSCI.1821-10.2010

Flotillin-1 is required for the Dynamin-independent, ERM-dependent endocytosis of Sema3A (A) Images show that Flotillin knockdown reduces the amount of internalized Sema3A (in black) compared to growth cones expressing control shRNAs. Growth cone boundaries are delineated by masks (green). Addition of dynasore 5 mins before and during incubation with Sema3A appears to decrease uptake. Together with Flotillin shRNA, dynasore more dramatically decreases Sema3A endocytosis. (B) Bar graph compares mean area internalized Sema3A per growth cone area, normalized to controls (n=34 growth cones from 2 experiments, p=0.0058, one-way ANOVA; * indicates p
Figure Legend Snippet: Flotillin-1 is required for the Dynamin-independent, ERM-dependent endocytosis of Sema3A (A) Images show that Flotillin knockdown reduces the amount of internalized Sema3A (in black) compared to growth cones expressing control shRNAs. Growth cone boundaries are delineated by masks (green). Addition of dynasore 5 mins before and during incubation with Sema3A appears to decrease uptake. Together with Flotillin shRNA, dynasore more dramatically decreases Sema3A endocytosis. (B) Bar graph compares mean area internalized Sema3A per growth cone area, normalized to controls (n=34 growth cones from 2 experiments, p=0.0058, one-way ANOVA; * indicates p

Techniques Used: Expressing, Incubation, shRNA

43) Product Images from "Acute dynamin inhibition dissects synaptic vesicle recycling pathways that drive spontaneous and evoked neurotransmission"

Article Title: Acute dynamin inhibition dissects synaptic vesicle recycling pathways that drive spontaneous and evoked neurotransmission

Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

doi: 10.1523/JNEUROSCI.3427-09.2010

Spontaneous and evoked transmission occur at the same synapses and are differentially affected by dynasore
Figure Legend Snippet: Spontaneous and evoked transmission occur at the same synapses and are differentially affected by dynasore

Techniques Used: Transmission Assay

Incubation with dynasore during stimulation causes vesicle depletion
Figure Legend Snippet: Incubation with dynasore during stimulation causes vesicle depletion

Techniques Used: Incubation

Prolonged dynasore application failed to suppress spontaneous neurotransmission
Figure Legend Snippet: Prolonged dynasore application failed to suppress spontaneous neurotransmission

Techniques Used:

44) Product Images from "Regulation of G-protein signaling via Gnas is required to regulate proximal tubular growth in the Xenopus pronephros"

Article Title: Regulation of G-protein signaling via Gnas is required to regulate proximal tubular growth in the Xenopus pronephros

Journal: Developmental biology

doi: 10.1016/j.ydbio.2013.01.017

Imbalance of endo- and exocytosis by Ctx causes proximal tubular defects. (A–J) Whole mount immunostaining with the 3G8 and 4A6 antibody of control embryos or embryos treated with 50 mM Dynasore in the presence or absence of 2 μg/ml Ctx or 50 μM Golgicide A at stage 40. (K–N) Bar diagrams quantifying proximal tubular cell numbers at stage 42 (K and M) and ectopic 4A6 staining at stage 40 (L and N) summarizing three independent experiments. Data were analyzed by Student’s t -test with indicating a p value of
Figure Legend Snippet: Imbalance of endo- and exocytosis by Ctx causes proximal tubular defects. (A–J) Whole mount immunostaining with the 3G8 and 4A6 antibody of control embryos or embryos treated with 50 mM Dynasore in the presence or absence of 2 μg/ml Ctx or 50 μM Golgicide A at stage 40. (K–N) Bar diagrams quantifying proximal tubular cell numbers at stage 42 (K and M) and ectopic 4A6 staining at stage 40 (L and N) summarizing three independent experiments. Data were analyzed by Student’s t -test with indicating a p value of

Techniques Used: Immunostaining, Staining

45) Product Images from "ADP-Ribosylation Factor 6 Mediates E-Cadherin Recovery by Chemical Chaperones"

Article Title: ADP-Ribosylation Factor 6 Mediates E-Cadherin Recovery by Chemical Chaperones

Journal: PLoS ONE

doi: 10.1371/journal.pone.0023188

Endocytosis inhibition increases E-cadherin at the plasma membrane. CHO cells stably transduced with the empty vector (Mock) or with WT, R749W or E757K hEcadherin were treated with Dynasore or MiTMAB for 17h. (A) Clathrin, E-cadherin and Arf6 expression were analyzed in whole cell lysates by Western Blot. Actin was used as a loading control. The intensity of the bands was quantified and normalized against the untreated WT cells. The graphs show the average + SE of protein level, in three independent experiments. (B) After treatment, Transferrin 594 was added to cells. Nucleus was counterstained with DAPI. The pictures were taken under a 63× objective. (C) Cells were fixed and immunostained with anti-human E-cadherin antibody. Nucleus was counterstained with DAPI. The pictures were taken under a 40× objective. (D) Flow cytometry technique was used to assess E-cadherin cell surface expression. Each histogram represents the cell surface expression of E-cadherin in cells WT, R749W or E757K, treated with Dynasore (red) or MiTMAB (green) or untreated (blue). The black area in the histogram represents the cells that were not incubated with primary antibody, this sample was used as negative control. For each sample, the number of cells expressing surface E-cadherin was calculated. The mean fluorescence intensity was also quantified and normalized against the control of WT expressing cells. The graphs show the average + SE, n = 3 (* represents p≤0.05).
Figure Legend Snippet: Endocytosis inhibition increases E-cadherin at the plasma membrane. CHO cells stably transduced with the empty vector (Mock) or with WT, R749W or E757K hEcadherin were treated with Dynasore or MiTMAB for 17h. (A) Clathrin, E-cadherin and Arf6 expression were analyzed in whole cell lysates by Western Blot. Actin was used as a loading control. The intensity of the bands was quantified and normalized against the untreated WT cells. The graphs show the average + SE of protein level, in three independent experiments. (B) After treatment, Transferrin 594 was added to cells. Nucleus was counterstained with DAPI. The pictures were taken under a 63× objective. (C) Cells were fixed and immunostained with anti-human E-cadherin antibody. Nucleus was counterstained with DAPI. The pictures were taken under a 40× objective. (D) Flow cytometry technique was used to assess E-cadherin cell surface expression. Each histogram represents the cell surface expression of E-cadherin in cells WT, R749W or E757K, treated with Dynasore (red) or MiTMAB (green) or untreated (blue). The black area in the histogram represents the cells that were not incubated with primary antibody, this sample was used as negative control. For each sample, the number of cells expressing surface E-cadherin was calculated. The mean fluorescence intensity was also quantified and normalized against the control of WT expressing cells. The graphs show the average + SE, n = 3 (* represents p≤0.05).

Techniques Used: Inhibition, Stable Transfection, Transduction, Plasmid Preparation, Expressing, Western Blot, Flow Cytometry, Cytometry, Incubation, Negative Control, Fluorescence

46) Product Images from "Constitutive and Chemokine-dependent Internalization and Recycling of CXCR7 in Breast Cancer Cells to Degrade Chemokine Ligands"

Article Title: Constitutive and Chemokine-dependent Internalization and Recycling of CXCR7 in Breast Cancer Cells to Degrade Chemokine Ligands

Journal: Oncogene

doi: 10.1038/onc.2010.212

Clathrin-mediated endocytosis and β-arrestin 2 regulate CXCR7-dependent accumulation of chemokines, receptor internalization, and total cell membrane CXCR7 (A) 231-CXCR7 (CXCR7) and 231-control (231) cells were incubated with 0.4M sucrose, 80 μM dynasore, or vehicle control for 30 minutes prior to incubation with ≈ 4 ng/ml CXCL12-GL for various periods of time. Amounts of intracellular chemokine were normalized to total protein per well and graphed as mean ± SEM at each time point (n = 4 per condition). *, p
Figure Legend Snippet: Clathrin-mediated endocytosis and β-arrestin 2 regulate CXCR7-dependent accumulation of chemokines, receptor internalization, and total cell membrane CXCR7 (A) 231-CXCR7 (CXCR7) and 231-control (231) cells were incubated with 0.4M sucrose, 80 μM dynasore, or vehicle control for 30 minutes prior to incubation with ≈ 4 ng/ml CXCL12-GL for various periods of time. Amounts of intracellular chemokine were normalized to total protein per well and graphed as mean ± SEM at each time point (n = 4 per condition). *, p

Techniques Used: Incubation

47) Product Images from "Calcitonin Gene-Related Peptide Induces HIV-1 Proteasomal Degradation in Mucosal Langerhans Cells"

Article Title: Calcitonin Gene-Related Peptide Induces HIV-1 Proteasomal Degradation in Mucosal Langerhans Cells

Journal: Journal of Virology

doi: 10.1128/JVI.01205-17

CGRP acts at a viral postentry step and directs HIV-1 for proteasomal degradation to inhibit trans -infection in MDLCs. (A) Untreated or CGRP-treated (24 h; 100 nM) MDLCs were pulsed with HIV-1 ADA at 37°C for 4 h in the absence or presence of either chloroquine (50 μM) or MG132 (10 μM), washed, treated with trypsin for 10 min to remove surface-bound virus, and lysed, and intracellular HIV-1 was measured in the cell lysates using p24 ELISA. Shown are means ± SEMs ( n = 3) for HIV-1 degradation indexes, calculated as the ratios of intracellular HIV-1 in the presence over absence of inhibitors. *, P = 0.0127 (untreated) and 0.0434 (CGRP treated) (MG132 versus chloroquine, respectively). (B to D) Untreated or CGRP-treated (24 h, 100 nM) MDLCs were pulsed with HIV-1 JR-CSF for 4 h at 37°C in the absence (B) or presence of the indicated concentrations of NH 4 Cl and lactacystin (C) or dynasore (D). The cells were then cocultured with autologous CD4 + T cells for a week. In panel B, the cells were collected, stained for surface CD1a (upper and middle portions) or CD3/CD4 (lower portion) followed by intracellular p24, and examined by flow cytometry. Shown are representative fluorescence-activated cell sorting (FACS) plots, with regular numbers indicating the means ± SEMs ( n = 3 for each panel) for percentages of p24 + cells out of FSC high SSC high CD1a + MDLCs (upper portion), FSC low SSC low CD1a − T cells (middle portion), or SSC low CD3 + CD4 + T cells (lower portion). P = 0.0083, 0.0005, and 0.0302 for CGRP-treated versus untreated CD1a + MDLCs, CD1a − T cells, or CD4 + T cells, respectively. Numbers in italics show means ± SEMs for mean fluorescence intensity (MFI) of CD4 expression (lower portion). FSC, forward scatter; SSC, side scatter. (C and D) HIV-1 replication was measured in the coculture supernatants by p24 ELISA. Shown are means ± SEMs for HIV-1 trans -infection percentages, normalized against untreated cells serving as the 100% set point, using MDLCs and T cells of 5 (C) or 3 (D) different donors.
Figure Legend Snippet: CGRP acts at a viral postentry step and directs HIV-1 for proteasomal degradation to inhibit trans -infection in MDLCs. (A) Untreated or CGRP-treated (24 h; 100 nM) MDLCs were pulsed with HIV-1 ADA at 37°C for 4 h in the absence or presence of either chloroquine (50 μM) or MG132 (10 μM), washed, treated with trypsin for 10 min to remove surface-bound virus, and lysed, and intracellular HIV-1 was measured in the cell lysates using p24 ELISA. Shown are means ± SEMs ( n = 3) for HIV-1 degradation indexes, calculated as the ratios of intracellular HIV-1 in the presence over absence of inhibitors. *, P = 0.0127 (untreated) and 0.0434 (CGRP treated) (MG132 versus chloroquine, respectively). (B to D) Untreated or CGRP-treated (24 h, 100 nM) MDLCs were pulsed with HIV-1 JR-CSF for 4 h at 37°C in the absence (B) or presence of the indicated concentrations of NH 4 Cl and lactacystin (C) or dynasore (D). The cells were then cocultured with autologous CD4 + T cells for a week. In panel B, the cells were collected, stained for surface CD1a (upper and middle portions) or CD3/CD4 (lower portion) followed by intracellular p24, and examined by flow cytometry. Shown are representative fluorescence-activated cell sorting (FACS) plots, with regular numbers indicating the means ± SEMs ( n = 3 for each panel) for percentages of p24 + cells out of FSC high SSC high CD1a + MDLCs (upper portion), FSC low SSC low CD1a − T cells (middle portion), or SSC low CD3 + CD4 + T cells (lower portion). P = 0.0083, 0.0005, and 0.0302 for CGRP-treated versus untreated CD1a + MDLCs, CD1a − T cells, or CD4 + T cells, respectively. Numbers in italics show means ± SEMs for mean fluorescence intensity (MFI) of CD4 expression (lower portion). FSC, forward scatter; SSC, side scatter. (C and D) HIV-1 replication was measured in the coculture supernatants by p24 ELISA. Shown are means ± SEMs for HIV-1 trans -infection percentages, normalized against untreated cells serving as the 100% set point, using MDLCs and T cells of 5 (C) or 3 (D) different donors.

Techniques Used: Infection, Enzyme-linked Immunosorbent Assay, Staining, Flow Cytometry, Cytometry, Fluorescence, FACS, Expressing

48) 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

49) 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

50) Product Images from "Design of a novel cell-permeable chimeric peptide to promote wound healing"

Article Title: Design of a novel cell-permeable chimeric peptide to promote wound healing

Journal: Scientific Reports

doi: 10.1038/s41598-018-34684-1

Effect of endocytosis inhibition on cellular uptake of Tylotoin-sC18* in HaCaT cells. ( A ) Cells were pretreated with 80 µM dynasore and subsequently with 10 µM CF-labeled Tylotoin-sC18* for further 30 min at 37 °C or cells were incubated with 10 µM CF-Tylotoin-sC18* for 30 min at 4 °C. green, CF-labeled peptide; blue, Hoechst 33342 nuclear stain; scale bar, 10 µm. ( B ) Corresponding flow cytometric uptake analysis of 10 µM CF-labeled peptide in HaCaT cells. (**p
Figure Legend Snippet: Effect of endocytosis inhibition on cellular uptake of Tylotoin-sC18* in HaCaT cells. ( A ) Cells were pretreated with 80 µM dynasore and subsequently with 10 µM CF-labeled Tylotoin-sC18* for further 30 min at 37 °C or cells were incubated with 10 µM CF-Tylotoin-sC18* for 30 min at 4 °C. green, CF-labeled peptide; blue, Hoechst 33342 nuclear stain; scale bar, 10 µm. ( B ) Corresponding flow cytometric uptake analysis of 10 µM CF-labeled peptide in HaCaT cells. (**p

Techniques Used: Inhibition, Labeling, Incubation, Staining, Flow Cytometry

51) Product Images from "Use of photoactivation and photobleaching to monitor the dynamic regulation of E-cadherin at the plasma membrane"

Article Title: Use of photoactivation and photobleaching to monitor the dynamic regulation of E-cadherin at the plasma membrane

Journal: Cell Adhesion & Migration

doi: 10.4161/cam.4.4.12661

Deregulation of intracellular trafficking specifically alters the rate of movement of E-cadherin at the plasma membrane. (A) Quantification of biotinylated E-cadherin internalization over 10 min in A431 cells or A431 cells treated with either dynasore or bafilomycin A1 (dynasore 80 µM, bafilomycin A1 1 µM, 30 min). (B) Fluorescence recovery curves following photobleaching of GFP-Farn2Palm (left part) and GFP-E-cadherin (right part) in control cells (blue) or cells treated with dynasore (red) or bafilomycin A1 (green), (dynasore 80 µM, bafilomycin A1 1 µM, 0.5–2 h). (C) Quantification of t 1/2 following photobleaching of GFP-Farn2Palm in control or dynasore-treated cells or GFP-E-cadherin in control cells or cells treated with either dynasore or bafilomycin A1. (D) Quantification of the speed of lateral movement of PAGFP-E-cadherin within the membrane in cells treated with bafilomycin A1. Values represent the mean from at least 25 cells. Error bars, s.e.m.
Figure Legend Snippet: Deregulation of intracellular trafficking specifically alters the rate of movement of E-cadherin at the plasma membrane. (A) Quantification of biotinylated E-cadherin internalization over 10 min in A431 cells or A431 cells treated with either dynasore or bafilomycin A1 (dynasore 80 µM, bafilomycin A1 1 µM, 30 min). (B) Fluorescence recovery curves following photobleaching of GFP-Farn2Palm (left part) and GFP-E-cadherin (right part) in control cells (blue) or cells treated with dynasore (red) or bafilomycin A1 (green), (dynasore 80 µM, bafilomycin A1 1 µM, 0.5–2 h). (C) Quantification of t 1/2 following photobleaching of GFP-Farn2Palm in control or dynasore-treated cells or GFP-E-cadherin in control cells or cells treated with either dynasore or bafilomycin A1. (D) Quantification of the speed of lateral movement of PAGFP-E-cadherin within the membrane in cells treated with bafilomycin A1. Values represent the mean from at least 25 cells. Error bars, s.e.m.

Techniques Used: Fluorescence

Inhibition of endocytosis strengthens cell-cell adhesion. (A) Representative images of fragments detached from a dispase-treated monolayer of control or dynasore (80 µM, 30 min) pre-treated A431 cells after mechanical stress. (B) Quantification of the number of single cells that disaggregate from a dispase-treated monolayer after mechanical stress. (C) Quantification of the number of single cells that disaggregate after mechanical stress from a dispase-treated monolayer of A431 or A431 GFP-E-cadherin cells, transfected with either control or E-cadherin siRNA. (D) Still images obtained from photobleaching experiments showing GFP-E-cadherin localization in A431 cells untreated or treated with dynasore (80 µM, 30 min). Scale bars, 20 µm. (E) Immunoblot showing surface and total levels of GFP- and endogenous E-cadherin (150 kDa and 120 kDa, respectively) in A431 cells untreated or treated with dynasore (80 µM, 30 min). (B and C) Values represent the mean from at least three independent experiments. Error bars, s.e.m.
Figure Legend Snippet: Inhibition of endocytosis strengthens cell-cell adhesion. (A) Representative images of fragments detached from a dispase-treated monolayer of control or dynasore (80 µM, 30 min) pre-treated A431 cells after mechanical stress. (B) Quantification of the number of single cells that disaggregate from a dispase-treated monolayer after mechanical stress. (C) Quantification of the number of single cells that disaggregate after mechanical stress from a dispase-treated monolayer of A431 or A431 GFP-E-cadherin cells, transfected with either control or E-cadherin siRNA. (D) Still images obtained from photobleaching experiments showing GFP-E-cadherin localization in A431 cells untreated or treated with dynasore (80 µM, 30 min). Scale bars, 20 µm. (E) Immunoblot showing surface and total levels of GFP- and endogenous E-cadherin (150 kDa and 120 kDa, respectively) in A431 cells untreated or treated with dynasore (80 µM, 30 min). (B and C) Values represent the mean from at least three independent experiments. Error bars, s.e.m.

Techniques Used: Inhibition, Transfection

The immobile fraction of E-cadherin at the plasma membrane is not perturbed by dynasore or bafilomycinA1. Quantification of immobile fraction following photobleaching of GFP-Farn2Palm in control or dynasore-treated cells or GFP-E-cadherin in control cells or cells treated with either dynasore or bafilomycin A1 (dynasore 80 µM, bafilomycin A1 1 µM, 0.5–2 h). Values represent the mean from at least 25 cells. Error bars, s.e.m.
Figure Legend Snippet: The immobile fraction of E-cadherin at the plasma membrane is not perturbed by dynasore or bafilomycinA1. Quantification of immobile fraction following photobleaching of GFP-Farn2Palm in control or dynasore-treated cells or GFP-E-cadherin in control cells or cells treated with either dynasore or bafilomycin A1 (dynasore 80 µM, bafilomycin A1 1 µM, 0.5–2 h). Values represent the mean from at least 25 cells. Error bars, s.e.m.

Techniques Used:

52) 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

53) Product Images from "Long-tip high-speed atomic force microscopy for nanometer-scale imaging in live cells"

Article Title: Long-tip high-speed atomic force microscopy for nanometer-scale imaging in live cells

Journal: Scientific Reports

doi: 10.1038/srep08724

Pits formation on the plasma membrane of a living COS-7 cell. (a) Fluorescence images of a COS-7 cells transfected with mEGFP. The white broken lines show the shadow of the cantilever. The area indicated with the white square was subjected to LT-HS-AFM imaging. (b) The number of observed pits per min per μm 2 area before, after and washout of dynasore application. (c) A sequence of magnified LT-HS-AFM images of a living COS-7 cell, taken at 6 s per frame, during the pit formation and the closure of the pit with a cap. Blue arrows indicate the formation of the pit. Dotted red circles indicate the formation of the closure cap. (d) A sequence of magnified LT-HS-AFM images during pit formation and closure of a pit without a cap. Blue arrows indicate the formation of the pit. (e) Time courses of the depth of pits with and without closure caps. More data are shown in Supplementary Figure 4 . Red bars indicate the formation of closure caps. Blue arrows indicate pit formation.
Figure Legend Snippet: Pits formation on the plasma membrane of a living COS-7 cell. (a) Fluorescence images of a COS-7 cells transfected with mEGFP. The white broken lines show the shadow of the cantilever. The area indicated with the white square was subjected to LT-HS-AFM imaging. (b) The number of observed pits per min per μm 2 area before, after and washout of dynasore application. (c) A sequence of magnified LT-HS-AFM images of a living COS-7 cell, taken at 6 s per frame, during the pit formation and the closure of the pit with a cap. Blue arrows indicate the formation of the pit. Dotted red circles indicate the formation of the closure cap. (d) A sequence of magnified LT-HS-AFM images during pit formation and closure of a pit without a cap. Blue arrows indicate the formation of the pit. (e) Time courses of the depth of pits with and without closure caps. More data are shown in Supplementary Figure 4 . Red bars indicate the formation of closure caps. Blue arrows indicate pit formation.

Techniques Used: Fluorescence, Transfection, Imaging, Sequencing

54) 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

55) Product Images from "Balancing spatially regulated β-actin translation and dynamin mediated endocytosis is required to assemble functional epithelial monolayers"

Article Title: Balancing spatially regulated β-actin translation and dynamin mediated endocytosis is required to assemble functional epithelial monolayers

Journal: Cytoskeleton (Hoboken, N.J.)

doi: 10.1002/cm.21265

Inhibiting dynamin mediated endocytosis with 80μM dynasore rescues the adherens junction assembly defects caused by partially mislocalizing β-actin translation
Figure Legend Snippet: Inhibiting dynamin mediated endocytosis with 80μM dynasore rescues the adherens junction assembly defects caused by partially mislocalizing β-actin translation

Techniques Used:

56) Product Images from "Astn2, A Novel Member of the Astrotactin Gene Family, Regulates the Trafficking of ASTN1 During Glial-Guided Neuronal Migration"

Article Title: Astn2, A Novel Member of the Astrotactin Gene Family, Regulates the Trafficking of ASTN1 During Glial-Guided Neuronal Migration

Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

doi: 10.1523/JNEUROSCI.0032-10.2010

The Dynamin inhibitor Dynasore inhibits glial-guided neuronal migration (A) A granule cell culture migration assay demonstrates that the effects of a soluble noncompetitive inhibitor of Dynamin, Dynasore on neuronal migration are reversible. Dissociated cerebellar granule neurons, labeled with a Venus encoding retrovirus, were imaged for three 60 minute periods; vehicle (DMSO) or DMSO and either 40 µM or 80 µM Dynasore were added during the second imaging period. To remove the Dynasore, we washed the cells three times with granule cell medium, and imaged migrating neurons as above for 1 hr. The images in (A) are inverted for better resolution and show migrating granule neurons labeled with Venus. In this study, we selected neurons with the features of actively migrating neurons, i.e. an elongated cell somata that was apposed to and flattened against Bergmann-like glial fibers. Migrating neurons are labeled by number in the 0 hr. panel and their final position is indicated by the same number in the 1 hr. panel. The number of cells that migrated more than 5 µm/hr were counted and expressed as a percentage of total labeled cells. (B) Cerebellar slices transfected with Venus expressing retrovirus were treated with Dynasore at 40 µM or 80 µM concentration, or with control vehicle (DMSO). TuJ1 staining reveals the parallel fiber layer of differentiated granule neurons in the inner EGL. In slices incubated with Dynasore, Venus expressing neurons seem to arrest in the EGL compared to DMSO control cells. DRAQ5 was used to counterstain nuclei. Quantitation of neurons in organotypic cerebellar slices shows that neurons remain significantly closer to the pial surface in the presence of 40 or 80 µM Dynasore, relative to controls (*p
Figure Legend Snippet: The Dynamin inhibitor Dynasore inhibits glial-guided neuronal migration (A) A granule cell culture migration assay demonstrates that the effects of a soluble noncompetitive inhibitor of Dynamin, Dynasore on neuronal migration are reversible. Dissociated cerebellar granule neurons, labeled with a Venus encoding retrovirus, were imaged for three 60 minute periods; vehicle (DMSO) or DMSO and either 40 µM or 80 µM Dynasore were added during the second imaging period. To remove the Dynasore, we washed the cells three times with granule cell medium, and imaged migrating neurons as above for 1 hr. The images in (A) are inverted for better resolution and show migrating granule neurons labeled with Venus. In this study, we selected neurons with the features of actively migrating neurons, i.e. an elongated cell somata that was apposed to and flattened against Bergmann-like glial fibers. Migrating neurons are labeled by number in the 0 hr. panel and their final position is indicated by the same number in the 1 hr. panel. The number of cells that migrated more than 5 µm/hr were counted and expressed as a percentage of total labeled cells. (B) Cerebellar slices transfected with Venus expressing retrovirus were treated with Dynasore at 40 µM or 80 µM concentration, or with control vehicle (DMSO). TuJ1 staining reveals the parallel fiber layer of differentiated granule neurons in the inner EGL. In slices incubated with Dynasore, Venus expressing neurons seem to arrest in the EGL compared to DMSO control cells. DRAQ5 was used to counterstain nuclei. Quantitation of neurons in organotypic cerebellar slices shows that neurons remain significantly closer to the pial surface in the presence of 40 or 80 µM Dynasore, relative to controls (*p

Techniques Used: Migration, Cell Culture, Labeling, Imaging, Transfection, Expressing, Concentration Assay, Staining, Incubation, Quantitation Assay

57) Product Images from "Complement membrane attack complexes activate noncanonical NF-κB by forming an Akt+NIK+ signalosome on Rab5+ endosomes"

Article Title: Complement membrane attack complexes activate noncanonical NF-κB by forming an Akt+NIK+ signalosome on Rab5+ endosomes

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

doi: 10.1073/pnas.1503535112

Dynasore inhibits noncanonical NF-κB–dependent Inflammatory genes in vitro and blocks clathrin-mediated endocytosis in vivo. HUVECs carrying an NF-κB–responsive luciferase reporter were pretreated for 30 min with vehicle,
Figure Legend Snippet: Dynasore inhibits noncanonical NF-κB–dependent Inflammatory genes in vitro and blocks clathrin-mediated endocytosis in vivo. HUVECs carrying an NF-κB–responsive luciferase reporter were pretreated for 30 min with vehicle,

Techniques Used: In Vitro, In Vivo, Luciferase

58) 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

59) 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

60) 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

61) Product Images from "Post-endocytic sorting of Plexin-D1 controls signal transduction and development of axonal and vascular circuits"

Article Title: Post-endocytic sorting of Plexin-D1 controls signal transduction and development of axonal and vascular circuits

Journal: Nature Communications

doi: 10.1038/ncomms14508

Sema3E induces Plexin-D1 endocytosis. ( a ) Examples of growth cones from E15.5 Pir neurons showing cell surface localization (Control) and internalization (+Sema3E) of VSV-Plexin-D1. ( b – e ) Quantification of the cell surface/total VSV-Plexin-D1 ratio in control growth cones and growth cones exposed to Sema3E (10 min of treatment) in the presence or absence of dynasore, Pitstop 2 or Pitstop 2-negative control. Sema3E induced clathrin- and dynamin-dependent internalization of Plexin-D1; n =number of growth cones analysed per condition in three independent experiments. Data are represented as mean±s.e.m., *** P
Figure Legend Snippet: Sema3E induces Plexin-D1 endocytosis. ( a ) Examples of growth cones from E15.5 Pir neurons showing cell surface localization (Control) and internalization (+Sema3E) of VSV-Plexin-D1. ( b – e ) Quantification of the cell surface/total VSV-Plexin-D1 ratio in control growth cones and growth cones exposed to Sema3E (10 min of treatment) in the presence or absence of dynasore, Pitstop 2 or Pitstop 2-negative control. Sema3E induced clathrin- and dynamin-dependent internalization of Plexin-D1; n =number of growth cones analysed per condition in three independent experiments. Data are represented as mean±s.e.m., *** P

Techniques Used: Negative Control

Endocytosis is required for Sema3E-induced growth cone collapse. ( a ) Collapse assay performed on E15.5 Pir neurons identified by tubulin in the presence or absence of Sema3E (20 min of treatment). Phalloidin staining shows the complex morphology of growth cones in the control condition and the collapsed morphology in the presence of Sema3E. ( b ) Image of growth cones of cultured E15.5 Pir neurons expressing clathrin light chain-CFP (CLC-CFP), with or without Sema3E (10 min of treatment). ( c – f ) Quantification of the percentage of collapsed growth cones in control cultures and in response to Sema3E (20 min of treatment). Sema3E-induced collapse was blocked by the endocytosis inhibitors dynasore and Pitstop 2; n =number of growth cones analysed per condition in three independent experiments. The χ 2 test, *** P
Figure Legend Snippet: Endocytosis is required for Sema3E-induced growth cone collapse. ( a ) Collapse assay performed on E15.5 Pir neurons identified by tubulin in the presence or absence of Sema3E (20 min of treatment). Phalloidin staining shows the complex morphology of growth cones in the control condition and the collapsed morphology in the presence of Sema3E. ( b ) Image of growth cones of cultured E15.5 Pir neurons expressing clathrin light chain-CFP (CLC-CFP), with or without Sema3E (10 min of treatment). ( c – f ) Quantification of the percentage of collapsed growth cones in control cultures and in response to Sema3E (20 min of treatment). Sema3E-induced collapse was blocked by the endocytosis inhibitors dynasore and Pitstop 2; n =number of growth cones analysed per condition in three independent experiments. The χ 2 test, *** P

Techniques Used: Staining, Cell Culture, Expressing

62) Product Images from "Omental Tissue-Mediated Tumorigenesis of Gastric Cancer Peritoneal Metastases"

Article Title: Omental Tissue-Mediated Tumorigenesis of Gastric Cancer Peritoneal Metastases

Journal: Frontiers in Oncology

doi: 10.3389/fonc.2019.01267

Characterization of human omental tissue-derived exosomes. (A) Exosomes were isolated from the CM of human omental tissue explants and analyzed by cryogenic transmission electron microscopy (Cryo-TEM); Scale bar = 100 nm. (B) Measurement of omental tissue exosomes diameter by nanoparticle tracking analysis system; (C) Western blot analysis of the exosome markers CD81 and CD63. Representatives of three independent experiments are shown; (D) PKH-67-labeled omental tissue exosomes were incubated with AGS gastric cancer cells, reaction was stopped at different time points (1, 3, 5, and 7 h) and cells were analyzed by confocal microscopy (upper panel). The cell's nucleus was stained with Dapi. PKH-67 labeled omental tissue exosomes were incubated for the indicated time points (30 min, 1, 2, 3, 4, 5, and 6 h) with AGS gastric cancer cells. Internalization was measured by flow cytometry (lower panel). Negative control-AGS cells with no addition of labeled exosomes. The data are presented as mean ±SD of four independent experiments; (E) AGS cells were pre-treated with 80 μM Dynasore or 10 ng/mL Heparin for 30 min or with 2 μM Simvastatin for 16 h. PKH-67 labeled omental tissue exosomes were incubated with untreated (positive control) or treated cells for 3 h and uptake was measured by flow cytometry analysis (right panel). The data are presented as the mean percent of cells that uptake labeled exosomes ± SD of three independent experiments; PKH-67 labeled omental tissue exosomes were incubated with untreated (positive control) or treated cells (Dynasore, Heparin or Simvastatin) and internalization was measured by confocal microscopy (left panel). Scale bar is 10 μm.
Figure Legend Snippet: Characterization of human omental tissue-derived exosomes. (A) Exosomes were isolated from the CM of human omental tissue explants and analyzed by cryogenic transmission electron microscopy (Cryo-TEM); Scale bar = 100 nm. (B) Measurement of omental tissue exosomes diameter by nanoparticle tracking analysis system; (C) Western blot analysis of the exosome markers CD81 and CD63. Representatives of three independent experiments are shown; (D) PKH-67-labeled omental tissue exosomes were incubated with AGS gastric cancer cells, reaction was stopped at different time points (1, 3, 5, and 7 h) and cells were analyzed by confocal microscopy (upper panel). The cell's nucleus was stained with Dapi. PKH-67 labeled omental tissue exosomes were incubated for the indicated time points (30 min, 1, 2, 3, 4, 5, and 6 h) with AGS gastric cancer cells. Internalization was measured by flow cytometry (lower panel). Negative control-AGS cells with no addition of labeled exosomes. The data are presented as mean ±SD of four independent experiments; (E) AGS cells were pre-treated with 80 μM Dynasore or 10 ng/mL Heparin for 30 min or with 2 μM Simvastatin for 16 h. PKH-67 labeled omental tissue exosomes were incubated with untreated (positive control) or treated cells for 3 h and uptake was measured by flow cytometry analysis (right panel). The data are presented as the mean percent of cells that uptake labeled exosomes ± SD of three independent experiments; PKH-67 labeled omental tissue exosomes were incubated with untreated (positive control) or treated cells (Dynasore, Heparin or Simvastatin) and internalization was measured by confocal microscopy (left panel). Scale bar is 10 μm.

Techniques Used: Derivative Assay, Isolation, Transmission Assay, Electron Microscopy, Transmission Electron Microscopy, Western Blot, Labeling, Incubation, Confocal Microscopy, Staining, Flow Cytometry, Cytometry, Negative Control, Positive Control

63) Product Images from "Dengue-3 Virus Entry into Vero Cells: Role of Clathrin-Mediated Endocytosis in the Outcome of Infection"

Article Title: Dengue-3 Virus Entry into Vero Cells: Role of Clathrin-Mediated Endocytosis in the Outcome of Infection

Journal: PLoS ONE

doi: 10.1371/journal.pone.0140824

DENV-3 entry into Vero cells is dependent on dynamin. (A) Cells were treated with dynasore and infected with DENV-3. After 1h of internalization in presence of the drug, monolayers were treated with proteinase K and the cell pellets were plated onto Vero cells to determine internalized virus by an infectious centre assay. (B) Cells treated with 150 μM dynasore or untreated (control) were infected with DENV-3. At 48 h p.i., immunofluorescence staining was carried out using mouse anti-E glycoprotein antibody. (C) Cells transiently transfected with GFP-Dyn II wt or GFP-Dyn II K44A were infected with DENV-3. After 24 h, cells were fixed and viral antigen expression was visualized by immunofluorescence staining using mouse anti-E glycoprotein antibody and TRITC-labelled anti-mouse IgG. (D) For quantification of samples shown in C, 250 transfected cells with similar levels of GFP expression were screened and cells positive for viral antigen were scored. In (A) and (D) results are expressed as the mean of three independent experiments ± SD. Asterisks indicate statistical significance (** p
Figure Legend Snippet: DENV-3 entry into Vero cells is dependent on dynamin. (A) Cells were treated with dynasore and infected with DENV-3. After 1h of internalization in presence of the drug, monolayers were treated with proteinase K and the cell pellets were plated onto Vero cells to determine internalized virus by an infectious centre assay. (B) Cells treated with 150 μM dynasore or untreated (control) were infected with DENV-3. At 48 h p.i., immunofluorescence staining was carried out using mouse anti-E glycoprotein antibody. (C) Cells transiently transfected with GFP-Dyn II wt or GFP-Dyn II K44A were infected with DENV-3. After 24 h, cells were fixed and viral antigen expression was visualized by immunofluorescence staining using mouse anti-E glycoprotein antibody and TRITC-labelled anti-mouse IgG. (D) For quantification of samples shown in C, 250 transfected cells with similar levels of GFP expression were screened and cells positive for viral antigen were scored. In (A) and (D) results are expressed as the mean of three independent experiments ± SD. Asterisks indicate statistical significance (** p

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

64) Product Images from "CD14 dependence of TLR4 endocytosis and TRIF signaling displays ligand specificity and is dissociable in endotoxin tolerance"

Article Title: CD14 dependence of TLR4 endocytosis and TRIF signaling displays ligand specificity and is dissociable in endotoxin tolerance

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

doi: 10.1073/pnas.1424980112

Dynasore inhibited LPS- and 1Z105-driven not UT12-induced TLR4 endocytosis but inhibited LPS-, UT12-, and 1Z105-induced IRF3 activation. BMDMs from WT and CD14 −/− mice were pretreated with either DMSO or dynasore (80 μM) for 60
Figure Legend Snippet: Dynasore inhibited LPS- and 1Z105-driven not UT12-induced TLR4 endocytosis but inhibited LPS-, UT12-, and 1Z105-induced IRF3 activation. BMDMs from WT and CD14 −/− mice were pretreated with either DMSO or dynasore (80 μM) for 60

Techniques Used: Activation Assay, Mouse Assay

65) Product Images from "ER contact sites define the position and timing of endosome fission"

Article Title: ER contact sites define the position and timing of endosome fission

Journal: Cell

doi: 10.1016/j.cell.2014.10.023

ER tubules define functional constrictions along dynasore-stalled tubular endosomes. (A) Images of live Cos-7 cells expressing mCh-Rab5 and GFP-Sec61β show that dynasore treatment elongates early endosomes while ER is unaffected. (B) Merged images
Figure Legend Snippet: ER tubules define functional constrictions along dynasore-stalled tubular endosomes. (A) Images of live Cos-7 cells expressing mCh-Rab5 and GFP-Sec61β show that dynasore treatment elongates early endosomes while ER is unaffected. (B) Merged images

Techniques Used: Functional Assay, Expressing

Dynasore-stalled tubular early endosomes undergo fission at ER contact sites. (A) The elongated tubular endosome phenotype that results from dynasore treatment can be reversed with the addition of FBS. Time-lapsed images of live Cos-7 cells expressing
Figure Legend Snippet: Dynasore-stalled tubular early endosomes undergo fission at ER contact sites. (A) The elongated tubular endosome phenotype that results from dynasore treatment can be reversed with the addition of FBS. Time-lapsed images of live Cos-7 cells expressing

Techniques Used: Expressing

66) 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

67) Product Images from "TLR9 is Actively Recruited to Aspergillus fumigatus Phagosomes and Requires the N-terminal Proteolytic Cleavage Domain for Proper Intracellular Trafficking"

Article Title: TLR9 is Actively Recruited to Aspergillus fumigatus Phagosomes and Requires the N-terminal Proteolytic Cleavage Domain for Proper Intracellular Trafficking

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi: 10.4049/jimmunol.1002760

Phagocytosis of Af conidia is dynamin-dependent and induces TLR9 recruitment within 45 min (A–C) Confocal microscopy of RAW macrophages expressing TLR9-GFP (green) and CD82-mRFP1 (red). One focal plane is shown. Scale bar is 5 μm. (A) The dynamin inhibitor Dynasore blocks uptake of Af conidia and polystyrene beads. RAW cells were pretreated with 100 mM Dynasore for 30 min before being exposed to Af resting conidia (unlabeled) and 1 μM fluorescent polystyrene beads (blue). The presence of Dynasore did not interfere with adherence of conidia and beads to the plasma membrane. (B) Af phagosomes acquire TLR9 and CD82 within 45 min after reversal of dynamin inhibition by Dynasore washout. Time-lapse images were acquired at indicated times DIC panel, white numbers in lower right corner indicate time (in min.) post washout. The white arrows in the red and green channels show the site of Af conidial entry. (C) Potent recruitment of TLR9 and CD82 to Af phagosomes is reached 1 h after Dynasore washout. The merged image on the right shows a representative cell having taken up five unlabeled Af resting conidia and one extracellular 1 μM polystyrene bead.
Figure Legend Snippet: Phagocytosis of Af conidia is dynamin-dependent and induces TLR9 recruitment within 45 min (A–C) Confocal microscopy of RAW macrophages expressing TLR9-GFP (green) and CD82-mRFP1 (red). One focal plane is shown. Scale bar is 5 μm. (A) The dynamin inhibitor Dynasore blocks uptake of Af conidia and polystyrene beads. RAW cells were pretreated with 100 mM Dynasore for 30 min before being exposed to Af resting conidia (unlabeled) and 1 μM fluorescent polystyrene beads (blue). The presence of Dynasore did not interfere with adherence of conidia and beads to the plasma membrane. (B) Af phagosomes acquire TLR9 and CD82 within 45 min after reversal of dynamin inhibition by Dynasore washout. Time-lapse images were acquired at indicated times DIC panel, white numbers in lower right corner indicate time (in min.) post washout. The white arrows in the red and green channels show the site of Af conidial entry. (C) Potent recruitment of TLR9 and CD82 to Af phagosomes is reached 1 h after Dynasore washout. The merged image on the right shows a representative cell having taken up five unlabeled Af resting conidia and one extracellular 1 μM polystyrene bead.

Techniques Used: Confocal Microscopy, Expressing, Inhibition

68) Product Images from "Profiling extracellular vesicle release by the filarial nematode Brugia malayi reveals sex-specific differences in cargo and a sensitivity to ivermectin"

Article Title: Profiling extracellular vesicle release by the filarial nematode Brugia malayi reveals sex-specific differences in cargo and a sensitivity to ivermectin

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0006438

Murine macrophages internalize parasite-derived EV by phagocytosis. Imaris 3D reconstructed confocal micrographs of murine J774A.1 macrophages. (A) Control macrophages showing internalization of PKH67-labeled EV (green) isolated from microfilaria (mf), L3, adult male (AM) and adult female (AF) worms in parallel with Fluoresbrite Carboxylate Microspheres (red, phagocytosis tracer). Macrophages are counterstained with Hoechst 33342 (nuclei, blue) and phalloidin (muscle, purple). (B) Macrophages treated with labeled EV (green) and microspheres (red) in the presence of 200 μM Dynasore. Absence of green and red indicates internalization of both EV and tracer are blocked. (C) Macrophages treated with labeled EV (green) and Alexa Fluor 555 conjugated transferrin (tracer, red) in the presence of 30 μM Chlorpromazine. Presence of green and absence of red indicates internalization of tracer is blocked but EV is not. (D) Macrophages treated with labeled EV (green) and Alexa Fluor 555 conjugated cholera toxin b (tracer, red) in the presence of 300 μM Genistein. Presence of green and general absence of red indicates internalization of tracer is generally blocked but EV is not. All Imaris images captured at magnification 68X, all scale bars 2 μm.
Figure Legend Snippet: Murine macrophages internalize parasite-derived EV by phagocytosis. Imaris 3D reconstructed confocal micrographs of murine J774A.1 macrophages. (A) Control macrophages showing internalization of PKH67-labeled EV (green) isolated from microfilaria (mf), L3, adult male (AM) and adult female (AF) worms in parallel with Fluoresbrite Carboxylate Microspheres (red, phagocytosis tracer). Macrophages are counterstained with Hoechst 33342 (nuclei, blue) and phalloidin (muscle, purple). (B) Macrophages treated with labeled EV (green) and microspheres (red) in the presence of 200 μM Dynasore. Absence of green and red indicates internalization of both EV and tracer are blocked. (C) Macrophages treated with labeled EV (green) and Alexa Fluor 555 conjugated transferrin (tracer, red) in the presence of 30 μM Chlorpromazine. Presence of green and absence of red indicates internalization of tracer is blocked but EV is not. (D) Macrophages treated with labeled EV (green) and Alexa Fluor 555 conjugated cholera toxin b (tracer, red) in the presence of 300 μM Genistein. Presence of green and general absence of red indicates internalization of tracer is generally blocked but EV is not. All Imaris images captured at magnification 68X, all scale bars 2 μm.

Techniques Used: Derivative Assay, Labeling, Isolation

69) Product Images from "Exosomes Derived from Epstein-Barr Virus-Infected Cells Are Internalized via Caveola-Dependent Endocytosis and Promote Phenotypic Modulation in Target Cells"

Article Title: Exosomes Derived from Epstein-Barr Virus-Infected Cells Are Internalized via Caveola-Dependent Endocytosis and Promote Phenotypic Modulation in Target Cells

Journal: Journal of Virology

doi: 10.1128/JVI.01310-13

Effect of endocytosis inhibitors on internalization of exosomes derived from Mutu cells into target cells. (A) Effect of endocytosis inhibitors on the internalization of DiI-exosomes. CNE1 cells were pretreated with DMSO, dynasore, or EIPA for 30 min
Figure Legend Snippet: Effect of endocytosis inhibitors on internalization of exosomes derived from Mutu cells into target cells. (A) Effect of endocytosis inhibitors on the internalization of DiI-exosomes. CNE1 cells were pretreated with DMSO, dynasore, or EIPA for 30 min

Techniques Used: Derivative Assay

70) Product Images from "Cell adhesion-mediated mitochondria transfer contributes to mesenchymal stem cell-induced chemoresistance on T cell acute lymphoblastic leukemia cells"

Article Title: Cell adhesion-mediated mitochondria transfer contributes to mesenchymal stem cell-induced chemoresistance on T cell acute lymphoblastic leukemia cells

Journal: Journal of Hematology & Oncology

doi: 10.1186/s13045-018-0554-z

Mitochondria transfer from Jurkat cells to MSCs can be blocked by cytochalasin D. a Representative confocal microscopy images show the presence of TNTs containing mitochondria (arrow). Scale bar, 5 μm. b 18-α-GA (50 μM), dynasore (50 μM), or cytochalasin D (1 μM) was added to the coculture system with ara-C or MTX for 48 h. Flow cytometry analysis of Jurkat cell-derived mitochondria uptake by MSCs (GFP+ gated). c The percentage of Red+ MSCs in each group was analyzed and graphed. The results are expressed as the mean ± S.E.M. of three independent experiments (* P
Figure Legend Snippet: Mitochondria transfer from Jurkat cells to MSCs can be blocked by cytochalasin D. a Representative confocal microscopy images show the presence of TNTs containing mitochondria (arrow). Scale bar, 5 μm. b 18-α-GA (50 μM), dynasore (50 μM), or cytochalasin D (1 μM) was added to the coculture system with ara-C or MTX for 48 h. Flow cytometry analysis of Jurkat cell-derived mitochondria uptake by MSCs (GFP+ gated). c The percentage of Red+ MSCs in each group was analyzed and graphed. The results are expressed as the mean ± S.E.M. of three independent experiments (* P

Techniques Used: Confocal Microscopy, Acetylene Reduction Assay, Flow Cytometry, Cytometry, Derivative Assay

71) Product Images from "Translocation of PEGylated quantum dots across rat alveolar epithelial cell monolayers"

Article Title: Translocation of PEGylated quantum dots across rat alveolar epithelial cell monolayers

Journal: International Journal of Nanomedicine

doi: 10.2147/IJN.S26051

Fluxes of quantum dots (6.25 μg/mL apical concentration) did not decrease in the presence of 80 μM dynasore for 24 hours. Transmonolayer resistance of rat alveolar epithelial cell monolayers was increased by about 30% after 24 hours of exposure to dynasore compared with control. Note: *Significantly increased from control (n = 6).
Figure Legend Snippet: Fluxes of quantum dots (6.25 μg/mL apical concentration) did not decrease in the presence of 80 μM dynasore for 24 hours. Transmonolayer resistance of rat alveolar epithelial cell monolayers was increased by about 30% after 24 hours of exposure to dynasore compared with control. Note: *Significantly increased from control (n = 6).

Techniques Used: Concentration Assay

72) Product Images from "Translocation of PEGylated quantum dots across rat alveolar epithelial cell monolayers"

Article Title: Translocation of PEGylated quantum dots across rat alveolar epithelial cell monolayers

Journal: International Journal of Nanomedicine

doi: 10.2147/IJN.S26051

Fluxes of quantum dots (6.25 μg/mL apical concentration) did not decrease in the presence of 80 μM dynasore for 24 hours. Transmonolayer resistance of rat alveolar epithelial cell monolayers was increased by about 30% after 24 hours of exposure to dynasore compared with control. Note: *Significantly increased from control (n = 6).
Figure Legend Snippet: Fluxes of quantum dots (6.25 μg/mL apical concentration) did not decrease in the presence of 80 μM dynasore for 24 hours. Transmonolayer resistance of rat alveolar epithelial cell monolayers was increased by about 30% after 24 hours of exposure to dynasore compared with control. Note: *Significantly increased from control (n = 6).

Techniques Used: Concentration Assay

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

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

Journal: American Journal of Respiratory Cell and Molecular Biology

doi: 10.1165/rcmb.2009-0138OC

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

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

74) Product Images from "The second extracellular loop dictates Occludin-mediated HCV entry"

Article Title: The second extracellular loop dictates Occludin-mediated HCV entry

Journal: Virology

doi: 10.1016/j.virol.2010.08.009

Dynasore has no adverse effect on cell viability. PHHs were treated with Dynasore (80μM) for 0, 1, 2, 4, 16, 24 hours. After removal of Dynasore, cells were incubated for additional 48 hours prior to the CellTiter- Glo cell viability assay (see
Figure Legend Snippet: Dynasore has no adverse effect on cell viability. PHHs were treated with Dynasore (80μM) for 0, 1, 2, 4, 16, 24 hours. After removal of Dynasore, cells were incubated for additional 48 hours prior to the CellTiter- Glo cell viability assay (see

Techniques Used: Incubation, Viability Assay

Dynasore treatment inhibits HCVpp entry into polarized primary human hepatocytes (PHHs). (A) Phase contrast image of PHHs (48 hours after culturing on collagen-coated plates). Bile canaliculi were visible as widened gaps between cells (white arrows).
Figure Legend Snippet: Dynasore treatment inhibits HCVpp entry into polarized primary human hepatocytes (PHHs). (A) Phase contrast image of PHHs (48 hours after culturing on collagen-coated plates). Bile canaliculi were visible as widened gaps between cells (white arrows).

Techniques Used:

Dynasore inhibits both HCVpp and HCVcc entry. (A) Schematic drawing of the experimental setup. Huh7.5.1 cells were treated with Dynasore or DMSO under four different protocols, differing in duration of the treatment and the time when added. A series of
Figure Legend Snippet: Dynasore inhibits both HCVpp and HCVcc entry. (A) Schematic drawing of the experimental setup. Huh7.5.1 cells were treated with Dynasore or DMSO under four different protocols, differing in duration of the treatment and the time when added. A series of

Techniques Used:

75) Product Images from "Clathrin-mediated post-fusion membrane retrieval influences the exocytic mode of endothelial Weibel-Palade bodies"

Article Title: Clathrin-mediated post-fusion membrane retrieval influences the exocytic mode of endothelial Weibel-Palade bodies

Journal: Journal of Cell Science

doi: 10.1242/jcs.200840

Cumulative fusion of WPBs results in improper release of VWF strings. (A,B) Control, mock, 25 μM Dynasore-treated or AP-2α knockdown HUVECs were stimulated with 100 ng/ml PMA. (A) The number of fusion events was monitored over a 10 min period by scanning confocal microscopy. There was no significant difference in the extent of fusion between Dynasore-treated and untreated cells ( n =13 control and drug-treated cells from 4 experiments). A significant difference in the amount of fusion was noted in AP-2α knockdown compared with mock, control and Dynasore-treated cells. Bars represent mean±s.e.m. ** P ≤0.01; ns, not significant, one-way ANOVA with Kruskall-Wallis post-test. (B) Treated, Dynasore-treated mock and AP-2α knockdown cells were incubated in the presence or absence of PMA (100 ng/ml) and the amount of VWF release determined by ELISA as a fraction of total VWF. Data are normalised to control and there is a small but significant inhibition of VWF release noted. Bars represent mean±s.e.m. ** P
Figure Legend Snippet: Cumulative fusion of WPBs results in improper release of VWF strings. (A,B) Control, mock, 25 μM Dynasore-treated or AP-2α knockdown HUVECs were stimulated with 100 ng/ml PMA. (A) The number of fusion events was monitored over a 10 min period by scanning confocal microscopy. There was no significant difference in the extent of fusion between Dynasore-treated and untreated cells ( n =13 control and drug-treated cells from 4 experiments). A significant difference in the amount of fusion was noted in AP-2α knockdown compared with mock, control and Dynasore-treated cells. Bars represent mean±s.e.m. ** P ≤0.01; ns, not significant, one-way ANOVA with Kruskall-Wallis post-test. (B) Treated, Dynasore-treated mock and AP-2α knockdown cells were incubated in the presence or absence of PMA (100 ng/ml) and the amount of VWF release determined by ELISA as a fraction of total VWF. Data are normalised to control and there is a small but significant inhibition of VWF release noted. Bars represent mean±s.e.m. ** P

Techniques Used: Confocal Microscopy, Incubation, Enzyme-linked Immunosorbent Assay, Inhibition

Inhibition of compensatory endocytosis increases the likelihood of cumulative exocytosis. (A-G) HUVECs were pre-incubated with control medium or 25 μM Dynasore-supplemented medium for 15 min before stimulation with 100 ng/ml PMA. (A,B) Cells were fixed after 10 min and prepared for TEM. Representative serial sections show multiple collapsed WPB (denoted by asterisks) with membranes touching projecting from the cell surface into the cytoplasm. Unfused WPBs (denoted by solid arrow heads) are also seen touching these collapsed structures. Open arrows show compensatory endocytic structures. The boxed area in Bii is shown at a higher magnification showing two clathrin-coated compensatory endocytic structures with wide necks. This likely reflects inhibited dynamin pinchase function. (C) Alexa Fluor 568-labelled transferrin was added to untreated, Dynasore-treated or AP-2α knockdown cells for 15 min. The mean fluorescence intensity of each cell over background was determined using ImageJ software (control, n =45 cells; Dynasore, n =40 cells; mock, n =69 cells; AP-2α, n =75 cells pooled from 4 experiments). (D-G) Control (E,F) or AP-2α knockdown (G) HUVECs were transfected with GFP-VWF and pre-incubated with (E,G) control medium or (F) 25 μM Dynasore-supplemented medium for 15 min, before stimulation with 100 ng/ml PMA and imaging for 10 min on a scanning confocal microscope. (D) Quantification of cumulative exocytosis in the presence and absence of 25 μM Dynasore ( n =13 control; n =13 Dynasore-treated cells; n =15 mock-treated cells; and n =19 AP-2α knockdown cells from 4 experiments). Representative images of (E) untreated cells at 0 and 265 s, (F) Dynasore-treated cells at 0 and 190 s post stimulation are shown. (G) AP-2α knockdown cells at 0 and 195 s, with the time in the top left representing the time of the first fusion event. Arrows indicate point of WPB fusion. (E) In control cells, despite fusion events occurring in close proximity, and within 80 s, the sites remain separate. (F) Fusion events occurring in close proximity and within 75 s in Dynasore-treated or within 125 s in AP-2α knockdown cells merge in what appears to be cumulative exocytosis. Bars represent mean±s.e.m. * P
Figure Legend Snippet: Inhibition of compensatory endocytosis increases the likelihood of cumulative exocytosis. (A-G) HUVECs were pre-incubated with control medium or 25 μM Dynasore-supplemented medium for 15 min before stimulation with 100 ng/ml PMA. (A,B) Cells were fixed after 10 min and prepared for TEM. Representative serial sections show multiple collapsed WPB (denoted by asterisks) with membranes touching projecting from the cell surface into the cytoplasm. Unfused WPBs (denoted by solid arrow heads) are also seen touching these collapsed structures. Open arrows show compensatory endocytic structures. The boxed area in Bii is shown at a higher magnification showing two clathrin-coated compensatory endocytic structures with wide necks. This likely reflects inhibited dynamin pinchase function. (C) Alexa Fluor 568-labelled transferrin was added to untreated, Dynasore-treated or AP-2α knockdown cells for 15 min. The mean fluorescence intensity of each cell over background was determined using ImageJ software (control, n =45 cells; Dynasore, n =40 cells; mock, n =69 cells; AP-2α, n =75 cells pooled from 4 experiments). (D-G) Control (E,F) or AP-2α knockdown (G) HUVECs were transfected with GFP-VWF and pre-incubated with (E,G) control medium or (F) 25 μM Dynasore-supplemented medium for 15 min, before stimulation with 100 ng/ml PMA and imaging for 10 min on a scanning confocal microscope. (D) Quantification of cumulative exocytosis in the presence and absence of 25 μM Dynasore ( n =13 control; n =13 Dynasore-treated cells; n =15 mock-treated cells; and n =19 AP-2α knockdown cells from 4 experiments). Representative images of (E) untreated cells at 0 and 265 s, (F) Dynasore-treated cells at 0 and 190 s post stimulation are shown. (G) AP-2α knockdown cells at 0 and 195 s, with the time in the top left representing the time of the first fusion event. Arrows indicate point of WPB fusion. (E) In control cells, despite fusion events occurring in close proximity, and within 80 s, the sites remain separate. (F) Fusion events occurring in close proximity and within 75 s in Dynasore-treated or within 125 s in AP-2α knockdown cells merge in what appears to be cumulative exocytosis. Bars represent mean±s.e.m. * P

Techniques Used: Inhibition, Incubation, Transmission Electron Microscopy, Fluorescence, Software, Transfection, Imaging, Microscopy

Related Articles

Immunocytochemistry:

Article Title: Endocytosis-dependent coordination of multiple actin regulators is required for wound healing
Article Snippet: Paragraph title: Cell culture, scratch wound assay, and immunocytochemistry ... Dynasore (EMD Millipore) was diluted to 100 mM stock in DMSO.

Whole Genome Amplification:

Article Title: Notch Promotes Dynamin-Dependent Endocytosis of Nephrin
Article Snippet: In some experiments, transfectants were incubated with Dynasore (160 μm; Sigma) or M β CD (10 mM; Sigma) for 30 minutes at 37°C. .. Fixed, permeabilized cells were co-incubated with anti-nephrin and anti-FLAG primary antibodies for 1 hour at room temperature, washed three times with PBS, and co-incubated for 1 hour at room temperature with AlexaFluor 488– and 594–conjugated secondary antibodies for anti-nephrin and anti-FLAG immunodetection, respectively, and AlexaFluor 647–conjugated WGA for membrane labeling.

Mass Spectrometry:

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)
Article Snippet: The peptide purity was > 95% as determined by Anaspec Inc. by MS and RP-HPLC. .. The endocytosis inhibitors cytochalasin A, cytochalasin D, dynasore, wortmannin, 1,1′-Disulfanediyldinaphthalen-2-ol (IPA-3), and chlorpromazine hydrochloride (CPZ) were from Sigma Aldrich (Stockholm, Sweden).

Positive Control:

Article Title: Knockdown of specific host factors protects against influenza virus-induced cell death
Article Snippet: .. Dynasore (Sigma Aldrich, Oakville, ON, Canada) dynamin inhibitor was used as a positive control. .. Cells were pretreated with 80 μ M Dynasore in the absence of serum for 1 h, and the inhibitor was included for all stages of infection thereafter.

Synthesized:

Article Title: Hippocampal Long-Term Depression in the Presence of Calcium-Permeable AMPA Receptors
Article Snippet: Peptides S3 (MASGVAVSDGVIKVFN) that inhibits cofilin phosphorylation and pS3 (MASpGVAVSDGVIKVFN) that inhibits cofilin dephosphorylation were synthesized by GenScript and the APTC peptide synthesis facility. .. Dynasore was purchased from Sigma Aldrich.

Article Title: Cathelicidin promotes inflammation by enabling binding of self-RNA to cell surface scavenger receptors
Article Snippet: Chemicals Synthetic cathelicidin peptides were synthesized and purchased from Genemed synthesis Inc (San Antonio, TX). .. MDC, dynasore, fucoidan, cycloheximide, Pertussis toxin from Bordetella pertussis, and saponin from quillaja bark were all purchased from Sigma-Aldrich (St Louis, MO).

Cytometry:

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
Article Snippet: Dynasore, a specific dynamin-mediated endocytic inhibitor (Macia et al., ), Methyl-β-cyclodextrin (MβCD), a membrane cholesterol extractor (Zidovetzki and Levitan, ), and Amiloride hydrochloride, a macropinocytosis inhibitor (Koivusalo et al., ) were purchased from Sigma Aldrich, St. Louis, MO, USA. .. A mouse monoclonal antibody against the A-subunit of Stx2 (Mab 2E11) was provided by Roxane Piazza (Butantan Institute, São Paulo, SP, Brazil) (Rocha et al., ) and an Alexa 647-conjugated anti-mouse secondary antibody (AbCam, catalog #ab150115) were used for flow cytometry.

Article Title: Maintenance of CD4 T cell fitness through regulation of Foxo1
Article Snippet: To measure cell proliferation and survival, sorted cells were labeled with CellTrace Violet (Invitrogen) before being cultured with plate-bound anti-CD3 and soluble anti-CD28; before flow cytometry, cells were stained with either 7AAAD (eBioscience) or Live/Dead Fixable Dead Cell stain. .. For inhibition of dynamin- and clathrin-mediated endocytosis, Dynasore (Sigma-Aldrich) was used.

Article Title: Knockdown of specific host factors protects against influenza virus-induced cell death
Article Snippet: Paragraph title: Flow cytometry ... Dynasore (Sigma Aldrich, Oakville, ON, Canada) dynamin inhibitor was used as a positive control.

Blocking Assay:

Article Title: Endocytosis-dependent coordination of multiple actin regulators is required for wound healing
Article Snippet: Dynasore (EMD Millipore) was diluted to 100 mM stock in DMSO. .. After being washed with PBS, the cells were incubated overnight with rabbit anti–phospho-myosin light chain 2 (Ser19) antibody (dilution 1:50; Cell Signaling Technology) or mouse anti–clathrin heavy chain antibody (dilution 1:1,000; Merck) in the blocking solution (PBS containing 3% BSA and 0.5% Triton X-100) at 4°C.

Article Title: Maintenance of CD4 T cell fitness through regulation of Foxo1
Article Snippet: For IL-2 withdrawal assays, anti-IL-2 blocking antibody (JES6–1A12, BioLegend) was used. .. For inhibition of dynamin- and clathrin-mediated endocytosis, Dynasore (Sigma-Aldrich) was used.

Adsorption:

Article Title: Knockdown of specific host factors protects against influenza virus-induced cell death
Article Snippet: After virus adsorption, cells were incubated at 35o C for 5 h. For intracellular antigen detection, cells were fixed with 70% ethanol (v/v), permeabilized with 0.25% Triton-X-100 and probed with mouse anti-NP mAb conjugated with Alexa Fluor 350 (Invitrogen). .. Dynasore (Sigma Aldrich, Oakville, ON, Canada) dynamin inhibitor was used as a positive control.

Real-time Polymerase Chain Reaction:

Article Title: Fusion of Anthopleurin-B to AAV2 increases specificity of cardiac gene transfer
Article Snippet: 1×106 H9C2, NRVM, HEK293, and HEK293-Nav 1.5(α) cells were plated into T12.5 flasks; after 24hs, media was discarded, cells were abundantly washed three times with chilled PBS, and incubated for 30 minutes in 1 ml of serum-free DMEM containing 80 μM of Dynasore (Sigma, St. Louis, MO) at 37 °C, 5% CO2, to arrest endocytosis. .. Total DNA was isolated and quantified by qPCR.

Incubation:

Article Title: Endocytosis-dependent coordination of multiple actin regulators is required for wound healing
Article Snippet: Dynasore (EMD Millipore) was diluted to 100 mM stock in DMSO. .. Cells were incubated in fresh culture medium containing control DMSO (0.24% vol/vol) or final 80 µM dynasore for 30 min before wounding.

Article Title: Endocytosis of Murine Norovirus 1 into Murine Macrophages Is Dependent on Dynamin II and Cholesterol ▿
Article Snippet: .. Cells were then incubated with the indicated concentrations of dynasore (Sigma-Aldrich, MO) in dimethyl sulfoxide (DMSO) or vehicle control for 30 min. .. Cells were infected with MNV-1 or VSV at the indicated MOI in the presence of dynasore or vehicle control for 60 min on ice.

Article Title: Notch Promotes Dynamin-Dependent Endocytosis of Nephrin
Article Snippet: .. In some experiments, transfectants were incubated with Dynasore (160 μm; Sigma) or M β CD (10 mM; Sigma) for 30 minutes at 37°C. ..

Article Title: Knockdown of specific host factors protects against influenza virus-induced cell death
Article Snippet: After virus adsorption, cells were incubated at 35o C for 5 h. For intracellular antigen detection, cells were fixed with 70% ethanol (v/v), permeabilized with 0.25% Triton-X-100 and probed with mouse anti-NP mAb conjugated with Alexa Fluor 350 (Invitrogen). .. Dynasore (Sigma Aldrich, Oakville, ON, Canada) dynamin inhibitor was used as a positive control.

Article Title: Fusion of Anthopleurin-B to AAV2 increases specificity of cardiac gene transfer
Article Snippet: .. 1×106 H9C2, NRVM, HEK293, and HEK293-Nav 1.5(α) cells were plated into T12.5 flasks; after 24hs, media was discarded, cells were abundantly washed three times with chilled PBS, and incubated for 30 minutes in 1 ml of serum-free DMEM containing 80 μM of Dynasore (Sigma, St. Louis, MO) at 37 °C, 5% CO2, to arrest endocytosis. .. Immediately after, 1×108 GC/ml of AAV vectors was added to the cells and incubated at 37 ºC, 5% CO2 for 60 minutes in Dynasore-containing media.

Cell Culture:

Article Title: Endocytosis-dependent coordination of multiple actin regulators is required for wound healing
Article Snippet: Paragraph title: Cell culture, scratch wound assay, and immunocytochemistry ... Dynasore (EMD Millipore) was diluted to 100 mM stock in DMSO.

Article Title: Maintenance of CD4 T cell fitness through regulation of Foxo1
Article Snippet: To measure cell proliferation and survival, sorted cells were labeled with CellTrace Violet (Invitrogen) before being cultured with plate-bound anti-CD3 and soluble anti-CD28; before flow cytometry, cells were stained with either 7AAAD (eBioscience) or Live/Dead Fixable Dead Cell stain. .. For inhibition of dynamin- and clathrin-mediated endocytosis, Dynasore (Sigma-Aldrich) was used.

Expressing:

Article Title: Notch Promotes Dynamin-Dependent Endocytosis of Nephrin
Article Snippet: Paragraph title: Nephrin Cell Surface Expression Assays in HEK293T cells ... In some experiments, transfectants were incubated with Dynasore (160 μm; Sigma) or M β CD (10 mM; Sigma) for 30 minutes at 37°C.

Filtration:

Article Title: Assessing mechanisms of GPIHBP1 and lipoprotein lipase movement across endothelial cells [S]
Article Snippet: Briefly, RHMVECs were grown on transwell filters (polyethylene terephthalate, 1.1-cm2 filtration area, 1 μm pore size; Millipore) that had been coated with 50 μg/ml fibronectin (BD Biosciences). .. In some experiments, cells were treated with genistein (Sigma), dynasore (Sigma), or vehicle (DMSO) alone.

Flow Cytometry:

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
Article Snippet: Dynasore, a specific dynamin-mediated endocytic inhibitor (Macia et al., ), Methyl-β-cyclodextrin (MβCD), a membrane cholesterol extractor (Zidovetzki and Levitan, ), and Amiloride hydrochloride, a macropinocytosis inhibitor (Koivusalo et al., ) were purchased from Sigma Aldrich, St. Louis, MO, USA. .. A mouse monoclonal antibody against the A-subunit of Stx2 (Mab 2E11) was provided by Roxane Piazza (Butantan Institute, São Paulo, SP, Brazil) (Rocha et al., ) and an Alexa 647-conjugated anti-mouse secondary antibody (AbCam, catalog #ab150115) were used for flow cytometry.

Article Title: Maintenance of CD4 T cell fitness through regulation of Foxo1
Article Snippet: To measure cell proliferation and survival, sorted cells were labeled with CellTrace Violet (Invitrogen) before being cultured with plate-bound anti-CD3 and soluble anti-CD28; before flow cytometry, cells were stained with either 7AAAD (eBioscience) or Live/Dead Fixable Dead Cell stain. .. For inhibition of dynamin- and clathrin-mediated endocytosis, Dynasore (Sigma-Aldrich) was used.

Article Title: Knockdown of specific host factors protects against influenza virus-induced cell death
Article Snippet: Paragraph title: Flow cytometry ... Dynasore (Sigma Aldrich, Oakville, ON, Canada) dynamin inhibitor was used as a positive control.

Transferring:

Article Title: Endocytosis-dependent coordination of multiple actin regulators is required for wound healing
Article Snippet: Dynasore (EMD Millipore) was diluted to 100 mM stock in DMSO. .. In the continuous presence of the drug, the cell sheets were scratch wounded with a pipette tip and fixed 0 and 15 min after wounding with 4% formaldehyde in PBS (room temperature 15 min).

Infection:

Article Title: Endocytosis of Murine Norovirus 1 into Murine Macrophages Is Dependent on Dynamin II and Cholesterol ▿
Article Snippet: Cells were then incubated with the indicated concentrations of dynasore (Sigma-Aldrich, MO) in dimethyl sulfoxide (DMSO) or vehicle control for 30 min. .. Cells were infected with MNV-1 or VSV at the indicated MOI in the presence of dynasore or vehicle control for 60 min on ice.

Article Title: Knockdown of specific host factors protects against influenza virus-induced cell death
Article Snippet: Dynasore (Sigma Aldrich, Oakville, ON, Canada) dynamin inhibitor was used as a positive control. .. Cells were pretreated with 80 μ M Dynasore in the absence of serum for 1 h, and the inhibitor was included for all stages of infection thereafter.

Indirect Immunoperoxidase Assay:

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)
Article Snippet: .. The endocytosis inhibitors cytochalasin A, cytochalasin D, dynasore, wortmannin, 1,1′-Disulfanediyldinaphthalen-2-ol (IPA-3), and chlorpromazine hydrochloride (CPZ) were from Sigma Aldrich (Stockholm, Sweden). .. The human neuroblastoma cell line SH-SY5Y was purchased from Sigma Aldrich, the Chinese hamster ovary cell line CHO-K1 was a kind gift from Prof. S. Lange, Gothenburg University and the murine fibroblast cell line NIH 3T3 was acquired from ATCC (Wezel, Germany).

Generated:

Article Title: Cathelicidin promotes inflammation by enabling binding of self-RNA to cell surface scavenger receptors
Article Snippet: U1 RNA and biotinylated U1 RNA were generated by in vitro transcription as described previously . .. MDC, dynasore, fucoidan, cycloheximide, Pertussis toxin from Bordetella pertussis, and saponin from quillaja bark were all purchased from Sigma-Aldrich (St Louis, MO).

Inhibition:

Article Title: Endocytosis of Murine Norovirus 1 into Murine Macrophages Is Dependent on Dynamin II and Cholesterol ▿
Article Snippet: Paragraph title: Growth curves (dynasore inhibition). ... Cells were then incubated with the indicated concentrations of dynasore (Sigma-Aldrich, MO) in dimethyl sulfoxide (DMSO) or vehicle control for 30 min.

Article Title: Maintenance of CD4 T cell fitness through regulation of Foxo1
Article Snippet: .. For inhibition of dynamin- and clathrin-mediated endocytosis, Dynasore (Sigma-Aldrich) was used. .. In vitro–cultured cells or cells isolated from lymphoid organs from mice were collected, resuspended in staining buffer (PBS containing 2% FBS) and stained.

Imaging:

Article Title: Quantifying the dynamic interactions between a clathrin-coated pit and cargo molecules
Article Snippet: .. Dynasore was dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich) and directly added to the imaging dish to a final concentration of 80 μM ( ). ..

Injection:

Article Title: Hsp70 downregulates dynamin in experimental stroke: a new therapeutic target?
Article Snippet: After control or OGD exposure, N2a cultures were treated with 60μM 17-AAG (Sigma) to induce Hsp70, or 30μM dynasore (Sigma), a dynamin inhibitor. .. Anesthetized mice were placed in a stereotaxic frame and a total volume 2μl of dynasore (0.3 mg/ml dose) or vehicle (0.1% DMF in PBS) was injected into the right lateral cerebral ventricle (stereotaxic coordinates: 1mm caudal to bregma, 1.3mm lateral to sagittal suture, and 2mm in depth) at a speed of 0.5μl/min via a burr hole.

Recombinant:

Article Title: CAR regulates epithelial cell junction stability through control of E-cadherin trafficking
Article Snippet: 8-pCPT-cGMP (PKG activator), H89 (PKA inhibitor) EIPA, PP2, PP3 and dynasore were obtained from Sigma-Aldrich. .. Ad5eGFP is E1-E3 deleted, replication deficient recombinant adenovirus type 5 that expresses green fluorescent protein under the control of the cytomegalovirus promoter (Baylor College of Medicine).

Molecular Weight:

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
Article Snippet: Dynasore, a specific dynamin-mediated endocytic inhibitor (Macia et al., ), Methyl-β-cyclodextrin (MβCD), a membrane cholesterol extractor (Zidovetzki and Levitan, ), and Amiloride hydrochloride, a macropinocytosis inhibitor (Koivusalo et al., ) were purchased from Sigma Aldrich, St. Louis, MO, USA. .. A fluorescein isothiocyanate (FITC)-labeled Dextran (average molecular weight of 70 kDa, Sigma Aldrich, catalog # 46945) was used as a marker of paracellular permeability (Chattopadhyay et al., ).

Plaque Assay:

Article Title: Endocytosis of Murine Norovirus 1 into Murine Macrophages Is Dependent on Dynamin II and Cholesterol ▿
Article Snippet: Cells were then incubated with the indicated concentrations of dynasore (Sigma-Aldrich, MO) in dimethyl sulfoxide (DMSO) or vehicle control for 30 min. .. Infection was allowed to proceed until the indicated time point, when the cells were freeze-thawed twice, and viral titers were determined by plaque assay as previously described ( ).

Isolation:

Article Title: Maintenance of CD4 T cell fitness through regulation of Foxo1
Article Snippet: Isolated CD4 T cells were stained with Live/Dead Fixable Dead Cell stain (Invitrogen) and the following fluorophoreconjugated antibodies from BioLegend: anti-CD4 (GK1.5), anti-CD25 (PC61), anti-CD44 (IM7) and anti-CD62L (MEL-14). .. For inhibition of dynamin- and clathrin-mediated endocytosis, Dynasore (Sigma-Aldrich) was used.

Article Title: Fusion of Anthopleurin-B to AAV2 increases specificity of cardiac gene transfer
Article Snippet: 1×106 H9C2, NRVM, HEK293, and HEK293-Nav 1.5(α) cells were plated into T12.5 flasks; after 24hs, media was discarded, cells were abundantly washed three times with chilled PBS, and incubated for 30 minutes in 1 ml of serum-free DMEM containing 80 μM of Dynasore (Sigma, St. Louis, MO) at 37 °C, 5% CO2, to arrest endocytosis. .. Total DNA was isolated and quantified by qPCR.

Transfection:

Article Title: Notch Promotes Dynamin-Dependent Endocytosis of Nephrin
Article Snippet: Briefly, HEK293T cells were plated onto glass coverslips at 50% confluence and either doubly transfected with p NephrinMYC and p NOTCH1-ICFLAG plasmids or singly transfected with p NephrinMYC plasmid alone. .. In some experiments, transfectants were incubated with Dynasore (160 μm; Sigma) or M β CD (10 mM; Sigma) for 30 minutes at 37°C.

Labeling:

Article Title: Maintenance of CD4 T cell fitness through regulation of Foxo1
Article Snippet: To measure cell proliferation and survival, sorted cells were labeled with CellTrace Violet (Invitrogen) before being cultured with plate-bound anti-CD3 and soluble anti-CD28; before flow cytometry, cells were stained with either 7AAAD (eBioscience) or Live/Dead Fixable Dead Cell stain. .. For inhibition of dynamin- and clathrin-mediated endocytosis, Dynasore (Sigma-Aldrich) was used.

Article Title: Notch Promotes Dynamin-Dependent Endocytosis of Nephrin
Article Snippet: In some experiments, transfectants were incubated with Dynasore (160 μm; Sigma) or M β CD (10 mM; Sigma) for 30 minutes at 37°C. .. Fixed, permeabilized cells were co-incubated with anti-nephrin and anti-FLAG primary antibodies for 1 hour at room temperature, washed three times with PBS, and co-incubated for 1 hour at room temperature with AlexaFluor 488– and 594–conjugated secondary antibodies for anti-nephrin and anti-FLAG immunodetection, respectively, and AlexaFluor 647–conjugated WGA for membrane labeling.

Article Title: Dynamin Is Required for GnRH Signaling to L-Type Calcium Channels and Activation of ERK
Article Snippet: All fluorescently labeled Alexa Fluor secondary antibodies were purchased from Molecular Probes-Life Technologies. .. GnRH, Buserelin (GnRHa), dynasore, and the anti-β-tubulin were purchased from Sigma.

Mouse Assay:

Article Title: Hippocampal Long-Term Depression in the Presence of Calcium-Permeable AMPA Receptors
Article Snippet: Paragraph title: Mice, Peptides and Chemicals ... Dynasore was purchased from Sigma Aldrich.

Article Title: Hsp70 downregulates dynamin in experimental stroke: a new therapeutic target?
Article Snippet: After control or OGD exposure, N2a cultures were treated with 60μM 17-AAG (Sigma) to induce Hsp70, or 30μM dynasore (Sigma), a dynamin inhibitor. .. Anesthetized mice were placed in a stereotaxic frame and a total volume 2μl of dynasore (0.3 mg/ml dose) or vehicle (0.1% DMF in PBS) was injected into the right lateral cerebral ventricle (stereotaxic coordinates: 1mm caudal to bregma, 1.3mm lateral to sagittal suture, and 2mm in depth) at a speed of 0.5μl/min via a burr hole.

Immunodetection:

Article Title: Notch Promotes Dynamin-Dependent Endocytosis of Nephrin
Article Snippet: In some experiments, transfectants were incubated with Dynasore (160 μm; Sigma) or M β CD (10 mM; Sigma) for 30 minutes at 37°C. .. Fixed, permeabilized cells were co-incubated with anti-nephrin and anti-FLAG primary antibodies for 1 hour at room temperature, washed three times with PBS, and co-incubated for 1 hour at room temperature with AlexaFluor 488– and 594–conjugated secondary antibodies for anti-nephrin and anti-FLAG immunodetection, respectively, and AlexaFluor 647–conjugated WGA for membrane labeling.

Microscopy:

Article Title: Notch Promotes Dynamin-Dependent Endocytosis of Nephrin
Article Snippet: In some experiments, transfectants were incubated with Dynasore (160 μm; Sigma) or M β CD (10 mM; Sigma) for 30 minutes at 37°C. .. After washes, coverslips were mounted on glass slides with DAKO fluorescent mounting medium and examined using a spinning disk Leica DMIRE2 confocal microscope with Hamamatsu back-thinned EM-CCD camera.

De-Phosphorylation Assay:

Article Title: Hippocampal Long-Term Depression in the Presence of Calcium-Permeable AMPA Receptors
Article Snippet: Peptides S3 (MASGVAVSDGVIKVFN) that inhibits cofilin phosphorylation and pS3 (MASpGVAVSDGVIKVFN) that inhibits cofilin dephosphorylation were synthesized by GenScript and the APTC peptide synthesis facility. .. Dynasore was purchased from Sigma Aldrich.

Staining:

Article Title: Maintenance of CD4 T cell fitness through regulation of Foxo1
Article Snippet: To measure cell proliferation and survival, sorted cells were labeled with CellTrace Violet (Invitrogen) before being cultured with plate-bound anti-CD3 and soluble anti-CD28; before flow cytometry, cells were stained with either 7AAAD (eBioscience) or Live/Dead Fixable Dead Cell stain. .. For inhibition of dynamin- and clathrin-mediated endocytosis, Dynasore (Sigma-Aldrich) was used.

Purification:

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
Article Snippet: Materials Purified Stx2a was provided from Phoenix Laboratory (Tufts Medical Center, Boston, MA, USA). .. Dynasore, a specific dynamin-mediated endocytic inhibitor (Macia et al., ), Methyl-β-cyclodextrin (MβCD), a membrane cholesterol extractor (Zidovetzki and Levitan, ), and Amiloride hydrochloride, a macropinocytosis inhibitor (Koivusalo et al., ) were purchased from Sigma Aldrich, St. Louis, MO, USA.

Article Title: CAR regulates epithelial cell junction stability through control of E-cadherin trafficking
Article Snippet: 8-pCPT-cGMP (PKG activator), H89 (PKA inhibitor) EIPA, PP2, PP3 and dynasore were obtained from Sigma-Aldrich. .. Ad5FK was produced and purified as previously described . p-CARthr290/ser293 polyclonal antibody was developed by Perbioscience (Thermofisher) using the peptide Ac-RTS(pT)AR(pS)YIGSNH-C and was affinity purified before use.

Affinity Purification:

Article Title: CAR regulates epithelial cell junction stability through control of E-cadherin trafficking
Article Snippet: 8-pCPT-cGMP (PKG activator), H89 (PKA inhibitor) EIPA, PP2, PP3 and dynasore were obtained from Sigma-Aldrich. .. Ad5FK was produced and purified as previously described . p-CARthr290/ser293 polyclonal antibody was developed by Perbioscience (Thermofisher) using the peptide Ac-RTS(pT)AR(pS)YIGSNH-C and was affinity purified before use.

Plasmid Preparation:

Article Title: Notch Promotes Dynamin-Dependent Endocytosis of Nephrin
Article Snippet: Briefly, HEK293T cells were plated onto glass coverslips at 50% confluence and either doubly transfected with p NephrinMYC and p NOTCH1-ICFLAG plasmids or singly transfected with p NephrinMYC plasmid alone. .. In some experiments, transfectants were incubated with Dynasore (160 μm; Sigma) or M β CD (10 mM; Sigma) for 30 minutes at 37°C.

Software:

Article Title: Knockdown of specific host factors protects against influenza virus-induced cell death
Article Snippet: Samples were analyzed with a Beckman Coulter MoFlo XDP Cell Sorter using Kaluza Analysis software (Beckman Coulter Canada, LP., Mississauga, ON, Canada). .. Dynasore (Sigma Aldrich, Oakville, ON, Canada) dynamin inhibitor was used as a positive control.

In Vitro:

Article Title: Maintenance of CD4 T cell fitness through regulation of Foxo1
Article Snippet: Paragraph title: CD4 enrichment, cell sorting and in vitro T cell stimulations. ... For inhibition of dynamin- and clathrin-mediated endocytosis, Dynasore (Sigma-Aldrich) was used.

Article Title: Cathelicidin promotes inflammation by enabling binding of self-RNA to cell surface scavenger receptors
Article Snippet: U1 RNA and biotinylated U1 RNA were generated by in vitro transcription as described previously . .. MDC, dynasore, fucoidan, cycloheximide, Pertussis toxin from Bordetella pertussis, and saponin from quillaja bark were all purchased from Sigma-Aldrich (St Louis, MO).

Produced:

Article Title: CAR regulates epithelial cell junction stability through control of E-cadherin trafficking
Article Snippet: 8-pCPT-cGMP (PKG activator), H89 (PKA inhibitor) EIPA, PP2, PP3 and dynasore were obtained from Sigma-Aldrich. .. Ad5FK was produced and purified as previously described . p-CARthr290/ser293 polyclonal antibody was developed by Perbioscience (Thermofisher) using the peptide Ac-RTS(pT)AR(pS)YIGSNH-C and was affinity purified before use.

Concentration Assay:

Article Title: Quantifying the dynamic interactions between a clathrin-coated pit and cargo molecules
Article Snippet: .. Dynasore was dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich) and directly added to the imaging dish to a final concentration of 80 μM ( ). ..

Scratch Wound Assay Assay:

Article Title: Endocytosis-dependent coordination of multiple actin regulators is required for wound healing
Article Snippet: Paragraph title: Cell culture, scratch wound assay, and immunocytochemistry ... Dynasore (EMD Millipore) was diluted to 100 mM stock in DMSO.

Marker:

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
Article Snippet: Dynasore, a specific dynamin-mediated endocytic inhibitor (Macia et al., ), Methyl-β-cyclodextrin (MβCD), a membrane cholesterol extractor (Zidovetzki and Levitan, ), and Amiloride hydrochloride, a macropinocytosis inhibitor (Koivusalo et al., ) were purchased from Sigma Aldrich, St. Louis, MO, USA. .. A fluorescein isothiocyanate (FITC)-labeled Dextran (average molecular weight of 70 kDa, Sigma Aldrich, catalog # 46945) was used as a marker of paracellular permeability (Chattopadhyay et al., ).

FACS:

Article Title: Maintenance of CD4 T cell fitness through regulation of Foxo1
Article Snippet: Paragraph title: CD4 enrichment, cell sorting and in vitro T cell stimulations. ... For inhibition of dynamin- and clathrin-mediated endocytosis, Dynasore (Sigma-Aldrich) was used.

Permeability:

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
Article Snippet: Dynasore, a specific dynamin-mediated endocytic inhibitor (Macia et al., ), Methyl-β-cyclodextrin (MβCD), a membrane cholesterol extractor (Zidovetzki and Levitan, ), and Amiloride hydrochloride, a macropinocytosis inhibitor (Koivusalo et al., ) were purchased from Sigma Aldrich, St. Louis, MO, USA. .. A fluorescein isothiocyanate (FITC)-labeled Dextran (average molecular weight of 70 kDa, Sigma Aldrich, catalog # 46945) was used as a marker of paracellular permeability (Chattopadhyay et al., ).

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  • 75
    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: 75/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dynamin inhibitory compounds dynasore/product/Millipore
    Average 75 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    dynamin inhibitory compounds dynasore - by Bioz Stars, 2020-02
    75/100 stars
      Buy from Supplier

    99
    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 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dynasore/product/Millipore
    Average 99 stars, based on 31 article reviews
    Price from $9.99 to $1999.99
    dynasore - by Bioz Stars, 2020-02
    99/100 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

    Dependence of HIV-derived ssRNA-induced foam cell formation and TNFα in macrophages on endocytosis and endosomal acidification. MDMs were pretreated with 100 µM chloroquine or 50 µM dynasore for 1 h followed by incubation with 1 µg/ml HIV-ssRNA for 24 h and stained with BODIPY and DAPI (A, C) and cell free supernatant analyzed for TNFα by ELISA and integrated fluorescence intensities for BODIPY in each cell of the confocal micrographs (B). Data shown is a representative experiment with similar results from four different healthy uninfected subjects. *p

    Journal: PLoS ONE

    Article Title: HIV-Derived ssRNA Binds to TLR8 to Induce Inflammation-Driven Macrophage Foam Cell Formation

    doi: 10.1371/journal.pone.0104039

    Figure Lengend Snippet: Dependence of HIV-derived ssRNA-induced foam cell formation and TNFα in macrophages on endocytosis and endosomal acidification. MDMs were pretreated with 100 µM chloroquine or 50 µM dynasore for 1 h followed by incubation with 1 µg/ml HIV-ssRNA for 24 h and stained with BODIPY and DAPI (A, C) and cell free supernatant analyzed for TNFα by ELISA and integrated fluorescence intensities for BODIPY in each cell of the confocal micrographs (B). Data shown is a representative experiment with similar results from four different healthy uninfected subjects. *p

    Article Snippet: Dynasore, and chloroquine were purchased from Calbiochem (San Diego, CA).

    Techniques: Derivative Assay, Incubation, Staining, Enzyme-linked Immunosorbent Assay, Fluorescence

    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