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  • 95
    Millipore dynasore hydrate
    Dynamin2 is essential for integrin-mediated static adhesion of human T cells. ( A - C ) Analysis of the adhesion of primary human resting CD4 + T cells to either ICAM-1-Fc or VCAM-1-Fc under static conditions. Lymphocytes were treated with DMSO as a control or 80μM <t>dynasore</t> to inhibit dynamin2 activity. If indicated, adhesion was stimulated with ( A , n = 3–5) 50ng/ml PMA, ( B , n = 3–5) 1μg/ml CXCL12 or ( C , n = 3) anti-CD3/CD28-coated beads (1:1 ratio to cells). 45min after seeding, the total number of adherent cells per mm 2 was quantified. ( D ) Detailed differential interference contrast (DIC) images of representative ICAM-1-Fc coated areas with adherent CD4 + T lymphocytes following treatment with DMSO or dynasore and, if indicated, stimulation with PMA or CXCL12. ( E , n = 4) Analysis of static adhesion of primary human resting CD4 + T cells to either ICAM-1-Fc or VCAM-1-Fc following treatment with dynole 31–2 as a control or dynole 34–2 to inhibit dynamin2 activity. If indicated, cells were stimulated with 50ng/ml PMA. 45min after seeding, the total number of adherent cells per mm 2 was quantified. ( F , n = 3) Analysis of static adhesion of primary human resting CD4 + T cells to either ICAM-1-Fc or VCAM-1-Fc 48h after the cells were transfected with either control siRNA or dynamin2 siRNA to knock down the large GTPase. If indicated, cells were stimulated with 50ng/ml PMA. 45min after seeding, the total number of adherent cells per mm 2 was quantified. Mean +SEM, *P≤0.05, **P≤0.01, ***P≤0.001.
    Dynasore Hydrate, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 213 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Millipore hydroxyl dynasore
    Inhibition of endocytic trafficking attenuates number and acidification of endolysosomes to halt autophagy flux. (a and b) Inhibition of endocytic trafficking in fibroblasts, with either <t>dynasore</t> or ciliobrevin A, consequently reduces the number of acidic vesicles indicated by LysoTracker Red fluorescence staining. Scale bar: 10 μm. Bar graphs in (b) represent the quantitative analysis of the LysoTracker Red fluorescence intensity and an average number of puncta per cell in both conditions. (c) Autophagy flux assay of HEK293T cells treated with DMSO and starved of amino acids for the indicated time points. Normal autophagy flux is indicated by the gradual decrease in the levels of LC3-II and SQSTM1 during starvation and their accumulation when the lysosomal function is inhibited by chloroquine. Bar graphs represent a quantitative analysis of the relative SQSTM1 and LC3 protein levels during starvation. (d) Autophagy flux was impaired in cells treated with either dynasore or ciliobrevin A. HEK293T Cells were pre-incubated with dynasore for 4 h or ciliobrevin A for 2 h prior to amino acid starvation for the indicated time points. Compared to controls (c), LC3-II protein levels did not decrease, but rather increased over time indicating an impaired autophagy flux due to lysosomal inhibition. Bar graphs represent a quantitative analysis of the relative SQSTM1 and LC3 protein levels during starvation. (e) Autophagic markers LC3 and SQSTM1 were decreased in cells overexpressing Flag-TFEB in an endocytic trafficking-dependent manner. Inhibition of dynein-dependent trafficking with ciliobrevin A strongly increased LC3-II and SQSTM1 protein levels. Bar graphs represent a quantitative analysis of the relative SQSTM1, LC3 and Flag-TFEB protein levels during starvation. A significant change relative to the control at the time point 0 h is given. Treatment with dynasore or ciliobrevin A did not affect fusion of autophagosomes with lysosomes (Figure S4). Data are represented as mean ± SEM, n = 6; ns denotes no significant difference, *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ANOVA.
    Hydroxyl Dynasore, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Millipore m dynasore
    Morphological changes upon combined <t>Dynasore-E2</t> treatment of MCF-7 cells. Semithin frozen sections of control MCF-7 cells labeled with antibodies directed against ER-α (green) and caveolin-1 (red). a – c ER-α receptor labeling occurred inside the cytoplasm and the plasma membrane in MCF-7 cells. Nuclear occurrence of the receptor could also be observed, however, to a lesser extent. The merged image of ER-α and caveolin-1 double labeling shows overlapping areas at the plasma membrane (arrowheads), while intracytoplasmic co-labeling could also be detected. Bars indicate 10 µm, nuclei were stained with DAPI. d Ultrathin cryosection shows morphologically distorted, elongated caveolae (arrowheads) indicating the effect of Dynasore treatment. Larger gold particles labeling ER-α could be observed in the close vicinity of caveolin-1 positive structures right beneath the plasma membrane (P). Note that the deeper cytoplasmic areas lack both small (ERα) and larger (caveolin-1) gold particles indicating the disturbed internalization of caveolin-1 positive vesicles. Bar indicates 200 nm
    M Dynasore, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    Millipore 80um dynasore
    Morphological changes upon combined <t>Dynasore-E2</t> treatment of MCF-7 cells. Semithin frozen sections of control MCF-7 cells labeled with antibodies directed against ER-α (green) and caveolin-1 (red). a – c ER-α receptor labeling occurred inside the cytoplasm and the plasma membrane in MCF-7 cells. Nuclear occurrence of the receptor could also be observed, however, to a lesser extent. The merged image of ER-α and caveolin-1 double labeling shows overlapping areas at the plasma membrane (arrowheads), while intracytoplasmic co-labeling could also be detected. Bars indicate 10 µm, nuclei were stained with DAPI. d Ultrathin cryosection shows morphologically distorted, elongated caveolae (arrowheads) indicating the effect of Dynasore treatment. Larger gold particles labeling ER-α could be observed in the close vicinity of caveolin-1 positive structures right beneath the plasma membrane (P). Note that the deeper cytoplasmic areas lack both small (ERα) and larger (caveolin-1) gold particles indicating the disturbed internalization of caveolin-1 positive vesicles. Bar indicates 200 nm
    80um Dynasore, supplied by Millipore, used in various techniques. Bioz Stars score: 77/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Millipore inhibitors dynasore
    A selective dynamin inhibitor <t>Dynasore</t> reduces βCTF and Aβ levels. A) N2a 695 cells were treated with dynasore at 10 µM or BACE Inhibitor IV at 15µM before subjected for further analysis. Levels of βCTF and Aβ were determined and significant reductions were observed upon dynasore or BACE inhibitor treatment, as compared to control (* p
    Inhibitors Dynasore, supplied by Millipore, used in various techniques. Bioz Stars score: 79/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Millipore epcs dynasore
    Blood flow measurement after injection of <t>EPCs,</t> EPCs + αPKCi, EPCs + <t>dynasore</t> groups (A) and the quantitative measurement of perfusion ratio of different groups at days 0, 7, 14, 28 respectively (B). *, P
    Epcs Dynasore, supplied by Millipore, used in various techniques. Bioz Stars score: 78/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    81
    Millipore huvec dynasore
    Blood flow measurement after injection of <t>EPCs,</t> EPCs + αPKCi, EPCs + <t>dynasore</t> groups (A) and the quantitative measurement of perfusion ratio of different groups at days 0, 7, 14, 28 respectively (B). *, P
    Huvec Dynasore, supplied by Millipore, used in various techniques. Bioz Stars score: 81/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Merck KGaA dynasore
    Entry of HCMV laboratory strains AD169 (BAD32) ( A ) and Towne ( B ) in HF upon treatment with pitstop 2, or <t>dynasore</t> as determined by expression of viral immediate early 1 (IE1) protein in infected cells. Confluent HF monolayers were pretreated with dynasore (100 μM) or pitstop 2 (25 μM) for 1 h, then infected with BAD32GFP or Towne virus at an MOI of 3.0 in the medium containing the same concentration of the drug for one hour, washed and thereafter incubated for 6 hours in the presence of the same concentration of drug before harvesting for immunoblots. Triplicate samples were used. DMSO-treated infected cells, untreated infected cells (UT) and untreated uninfected cells (UI) served as controls in this experiment. β-actin was used as a loading control. Immunoblots were digitally cropped to conserve the space.
    Dynasore, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 89/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Millipore dnm specific inhibitor dynasore
    Inhibition of <t>DNM</t> decreases nitrite production. A : nitrite production from COS7 cells overexpressing NOS1α + DNM2 was inhibited by the dynamin inhibitor <t>dynasore</t> in the presence and absence of ionomycin. Statistical analysis by 2-way ANOVA. *
    Dnm Specific Inhibitor Dynasore, supplied by Millipore, used in various techniques. Bioz Stars score: 78/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Millipore endocytosis inhibitor dynasore
    Osteoblast differentiation is arrested by inhibition of dynamin-dependent endocytosis. ( A,B ) Confluent C2C12 cells were serum-starved for 2 h prior to stimulation with the indicated concentrations of BMP-2. After 72 h of stimulation, cells were lysed and ALP activity was measured at 405 nm by conversion of para-nitrophenylphosphate. A schematic representation of the treatment is depicted above the respective histogram. The histograms show mean ± s.d. of triplicate measurements representative of three independent experiments. ( A ) During starvation and initial 4 h of stimulation, 40 µM <t>dynasore</t> or 0.05% DSMO were added. (P-values in relation to control treated samples). ( B ) After 4 h of stimulation, BMP-2-containing medium was replaced by medium without BMP-2. (** P-values in relation to unstimulated samples; 30 nM: P = 0.008, 60 nM: P = 0.0017) ( C–G ) Confluent C2C12 cells were treated as described in (A) with 30 nM BMP-2. Total RNA was isolated, cDNA was synthesized and mRNA expression was analyzed by qPCR using specific mouse primers ( Table S1 ). Histograms show mean normalized expression (MNE) with standard error of duplicate measurements relative to the housekeeping gene GAPDH . The results are representative of three independent experiments.
    Endocytosis Inhibitor Dynasore, supplied by Millipore, used in various techniques. Bioz Stars score: 79/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Millipore dynamin inhibitor dynasore
    Macrophage endocytosis of HMGB1 is mediated by RAGE-dependent pathway. ( a ) Confocal microscopy of alveolar macrophages (AMs) that were isolated from C57BL/6 (wild type, WT) mice and incubated with recombinant protein HMGB1-EGFP (100 nmol/l) or EGFP for 0–30 min. Original magnification × 600; results are representative of three independent experiments. ( b ) Confocal microscopy of AMs that were isolated from WT mice and incubated with 0–100 nmol/l HMGB1-EGFP for 30 min. Original magnification × 600; results are representative of three independent experiments. ( c ) Confocal microscopy of AMs that were isolated from WT, TLR2 −/− , TLR4 −/− , or RAGE −/− mice and incubated with HMGB1-EGFP (100 nmol/l) for 30 min. Original magnification × 600; results are representative of three independent experiments. ( d ) Changes in average number of intracellular EGFP-tagged protein particles in AMs, which were calculated using confocal microscopy program. The AMs were isolated from WT, TLR2 −/− , TLR4 −/− , or RAGE −/− mice and incubated with HMGB1-EGFP (100 nmol/l) for 30 min. In some experiments, WT AMs were incubated with HMGB1-EGFP and <t>dynamin</t> inhibitor <t>dynasore</t> (30 μ g/ml) for 30 min. The graph shows the mean and S.D., n =9. * P
    Dynamin Inhibitor Dynasore, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 67 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore gtpase inhibitor dynasore
    Macrophage endocytosis of HMGB1 is mediated by RAGE-dependent pathway. ( a ) Confocal microscopy of alveolar macrophages (AMs) that were isolated from C57BL/6 (wild type, WT) mice and incubated with recombinant protein HMGB1-EGFP (100 nmol/l) or EGFP for 0–30 min. Original magnification × 600; results are representative of three independent experiments. ( b ) Confocal microscopy of AMs that were isolated from WT mice and incubated with 0–100 nmol/l HMGB1-EGFP for 30 min. Original magnification × 600; results are representative of three independent experiments. ( c ) Confocal microscopy of AMs that were isolated from WT, TLR2 −/− , TLR4 −/− , or RAGE −/− mice and incubated with HMGB1-EGFP (100 nmol/l) for 30 min. Original magnification × 600; results are representative of three independent experiments. ( d ) Changes in average number of intracellular EGFP-tagged protein particles in AMs, which were calculated using confocal microscopy program. The AMs were isolated from WT, TLR2 −/− , TLR4 −/− , or RAGE −/− mice and incubated with HMGB1-EGFP (100 nmol/l) for 30 min. In some experiments, WT AMs were incubated with HMGB1-EGFP and <t>dynamin</t> inhibitor <t>dynasore</t> (30 μ g/ml) for 30 min. The graph shows the mean and S.D., n =9. * P
    Gtpase Inhibitor Dynasore, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    Millipore dynamin inhibitor dynasore monohydrate
    Macrophage endocytosis of HMGB1 is mediated by RAGE-dependent pathway. ( a ) Confocal microscopy of alveolar macrophages (AMs) that were isolated from C57BL/6 (wild type, WT) mice and incubated with recombinant protein HMGB1-EGFP (100 nmol/l) or EGFP for 0–30 min. Original magnification × 600; results are representative of three independent experiments. ( b ) Confocal microscopy of AMs that were isolated from WT mice and incubated with 0–100 nmol/l HMGB1-EGFP for 30 min. Original magnification × 600; results are representative of three independent experiments. ( c ) Confocal microscopy of AMs that were isolated from WT, TLR2 −/− , TLR4 −/− , or RAGE −/− mice and incubated with HMGB1-EGFP (100 nmol/l) for 30 min. Original magnification × 600; results are representative of three independent experiments. ( d ) Changes in average number of intracellular EGFP-tagged protein particles in AMs, which were calculated using confocal microscopy program. The AMs were isolated from WT, TLR2 −/− , TLR4 −/− , or RAGE −/− mice and incubated with HMGB1-EGFP (100 nmol/l) for 30 min. In some experiments, WT AMs were incubated with HMGB1-EGFP and <t>dynamin</t> inhibitor <t>dynasore</t> (30 μ g/ml) for 30 min. The graph shows the mean and S.D., n =9. * P
    Dynamin Inhibitor Dynasore Monohydrate, supplied by Millipore, used in various techniques. Bioz Stars score: 77/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    Millipore dynamin dynasore
    Macrophage endocytosis of HMGB1 is mediated by RAGE-dependent pathway. ( a ) Confocal microscopy of alveolar macrophages (AMs) that were isolated from C57BL/6 (wild type, WT) mice and incubated with recombinant protein HMGB1-EGFP (100 nmol/l) or EGFP for 0–30 min. Original magnification × 600; results are representative of three independent experiments. ( b ) Confocal microscopy of AMs that were isolated from WT mice and incubated with 0–100 nmol/l HMGB1-EGFP for 30 min. Original magnification × 600; results are representative of three independent experiments. ( c ) Confocal microscopy of AMs that were isolated from WT, TLR2 −/− , TLR4 −/− , or RAGE −/− mice and incubated with HMGB1-EGFP (100 nmol/l) for 30 min. Original magnification × 600; results are representative of three independent experiments. ( d ) Changes in average number of intracellular EGFP-tagged protein particles in AMs, which were calculated using confocal microscopy program. The AMs were isolated from WT, TLR2 −/− , TLR4 −/− , or RAGE −/− mice and incubated with HMGB1-EGFP (100 nmol/l) for 30 min. In some experiments, WT AMs were incubated with HMGB1-EGFP and <t>dynamin</t> inhibitor <t>dynasore</t> (30 μ g/ml) for 30 min. The graph shows the mean and S.D., n =9. * P
    Dynamin Dynasore, supplied by Millipore, used in various techniques. Bioz Stars score: 77/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Millipore caveolin dependent endocytosis inhibitor dynasore
    Macrophage endocytosis of HMGB1 is mediated by RAGE-dependent pathway. ( a ) Confocal microscopy of alveolar macrophages (AMs) that were isolated from C57BL/6 (wild type, WT) mice and incubated with recombinant protein HMGB1-EGFP (100 nmol/l) or EGFP for 0–30 min. Original magnification × 600; results are representative of three independent experiments. ( b ) Confocal microscopy of AMs that were isolated from WT mice and incubated with 0–100 nmol/l HMGB1-EGFP for 30 min. Original magnification × 600; results are representative of three independent experiments. ( c ) Confocal microscopy of AMs that were isolated from WT, TLR2 −/− , TLR4 −/− , or RAGE −/− mice and incubated with HMGB1-EGFP (100 nmol/l) for 30 min. Original magnification × 600; results are representative of three independent experiments. ( d ) Changes in average number of intracellular EGFP-tagged protein particles in AMs, which were calculated using confocal microscopy program. The AMs were isolated from WT, TLR2 −/− , TLR4 −/− , or RAGE −/− mice and incubated with HMGB1-EGFP (100 nmol/l) for 30 min. In some experiments, WT AMs were incubated with HMGB1-EGFP and <t>dynamin</t> inhibitor <t>dynasore</t> (30 μ g/ml) for 30 min. The graph shows the mean and S.D., n =9. * P
    Caveolin Dependent Endocytosis Inhibitor Dynasore, supplied by Millipore, used in various techniques. Bioz Stars score: 78/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Millipore gtpase dynamin inhibitor dynasore
    Tat induces TLR4 down modulation by specific <t>dynamin</t> endocytosis. ( A ) U937, HEK-TLR4-MD2-CD14 or human monocytes were stimulated or not with 10 or 100 nM of GST +/-Tat for the indicated times. Surface expression of TLR4 in unstimulated (black line) or stimulated (pink, blue and red lines) cells was analyzed by flow cytometry using anti-TLR4 or isotype control IgG (tinted), stained with secondary FITC or PE antibodies. Data represent one of three independent experiments. ( B ) U937 were pre-incubated for 1 h with 1 μg/mL of blocking anti-TLR4 or ( C ) HEK TLR4-CD14-MD2 were pretreated with 80 μM of <t>dynasore</t> for 30 minutes before stimulation with GST+/-Tat (100 nM) for the time periods indicated. Cell surface expression of TLR4 was then analyzed as described above. ( D – G ) The integrity of the raft structure is necessary for Tat-induced IL-6 and IL-8: D-E ) Purified human monocytes (10 6 ) were pretreated with increasing amounts of raft disruption drug M-β-CD (10, 60 min) or F-G ) with dynasore for 30 minutes before stimulation with GST-Tat 1–101 (100 nM). After 24 h, IL-6 and IL-8 production in the culture supernatants were quantified by ELISA. The data represent means (pg/ml) and SD (n > 3). As controls, cells were stimulated with PBS, DMSO or the highest drug concentration and cytokine production and cytotoxicity (trypan blue) were analyzed. Asterisks represent P values: *, P ˂ 0.05; **, P ˂ 0.01; ***, P ˂ 0.001, ns non significant.
    Gtpase Dynamin Inhibitor Dynasore, supplied by Millipore, used in various techniques. Bioz Stars score: 78/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Millipore endocytosis chemical inhibitors dynasore
    TuMV proteins VPg and CI localize to post-Golgi compartments. (A, B) Colocalization assay of TuMV fusion proteins with the early endosome marker mCherry-AtSYP61 (A) and the late endosome marker mCherry-AtARA6 (B) in Arabidopsis protoplasts. Images were taken at 20 hpt. (C, D) Effect of <t>dynasore</t> treatment on the subcellular localization of YFP-TuMV VPg (C) and YFP-TuMV CI (D). Protoplasts expressing YFP-TuMV VPg or YFP-TuMV CI at 16 hpt were treated with 100 µM dynasore or DMSO for 3 h and then 40 µM FM4-64 for 1 h. Scale bar = 10 μm.
    Endocytosis Chemical Inhibitors Dynasore, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    Millipore camello 5ht2a control experiments dynasore
    Optogenetic control and visualization of Ca 2+ signals with <t>CaMello-XRs.</t> a CaMello and CaMello-5HT 2A design. Both chimeric constructs consist of mouse melanopsin (mOpn4L), mCherry inserted into C3 and GCaMP6m added to the CT. CaMello-5HT 2A additionally has the 5-HT 2A receptor CT appended (C: intracellular loop; CT: C-terminus; H: transmembrane helix; E: extracellular loop). b Time course of light-induced Ca 2+ responses in HEK tsA201 cells. Transfected cells were visualized (mCherry, 561 nm) and Ca 2+ signals were measured (GCaMP6m, 476 + 495 nm) (images). Normalized Ca 2+ responses during 60 s of illumination (graphs), for CaMello-5HT 2A with/without addition of dynamin inhibitor <t>Dynasore</t> (50 µM) (mean ± s.e.m.; n = 5 dishes). Scale bar, 10 µm. c Normalized activation-dependent receptor internalization monitored via differences in membrane-localized mCherry fluorescence reduction between stimulated (476 nm + 561 nm) and unstimulated (561 nm) trials, for <t>CaMello-5HT2A</t> with/without addition of Dynasore (50 µM) (mean; n = 5 dishes). d Colocalization of CaMello-5HT 2A with Rab5a (-mCitrine, early endosome), Rab7a (early to late endosome) or GALT (beta-1,4-galactosyltransferase 1, trans-Golgi network) 5 min post stimulation with 476 nm light (5 min). Scale bar, 10 µm. e Averages of the calculated Pearson’s correlation coefficient for the colocalization as shown in d (box plot; one-way analysis of variance (ANOVA) and Holm-Sidak multiple comparison method; n = 6 individual cells for each colocalization pairing; *** p
    Camello 5ht2a Control Experiments Dynasore, supplied by Millipore, used in various techniques. Bioz Stars score: 87/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Millipore pharmacological compounds dynasore
    Optogenetic control and visualization of Ca 2+ signals with <t>CaMello-XRs.</t> a CaMello and CaMello-5HT 2A design. Both chimeric constructs consist of mouse melanopsin (mOpn4L), mCherry inserted into C3 and GCaMP6m added to the CT. CaMello-5HT 2A additionally has the 5-HT 2A receptor CT appended (C: intracellular loop; CT: C-terminus; H: transmembrane helix; E: extracellular loop). b Time course of light-induced Ca 2+ responses in HEK tsA201 cells. Transfected cells were visualized (mCherry, 561 nm) and Ca 2+ signals were measured (GCaMP6m, 476 + 495 nm) (images). Normalized Ca 2+ responses during 60 s of illumination (graphs), for CaMello-5HT 2A with/without addition of dynamin inhibitor <t>Dynasore</t> (50 µM) (mean ± s.e.m.; n = 5 dishes). Scale bar, 10 µm. c Normalized activation-dependent receptor internalization monitored via differences in membrane-localized mCherry fluorescence reduction between stimulated (476 nm + 561 nm) and unstimulated (561 nm) trials, for <t>CaMello-5HT2A</t> with/without addition of Dynasore (50 µM) (mean; n = 5 dishes). d Colocalization of CaMello-5HT 2A with Rab5a (-mCitrine, early endosome), Rab7a (early to late endosome) or GALT (beta-1,4-galactosyltransferase 1, trans-Golgi network) 5 min post stimulation with 476 nm light (5 min). Scale bar, 10 µm. e Averages of the calculated Pearson’s correlation coefficient for the colocalization as shown in d (box plot; one-way analysis of variance (ANOVA) and Holm-Sidak multiple comparison method; n = 6 individual cells for each colocalization pairing; *** p
    Pharmacological Compounds Dynasore, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Different effects of <t>dynasore</t> on the replication of HIV-1(VSV-G) and Wt in a human T cell, Rev-CEM. (A) Rev-CEM, a Rev-dependent GFP indicator cell, was pretreated for 30 minutes with 80 µM, 8 µM, or 0.8 µM dynasore, respectively, or treated with 0.1% DMSO as a control. Cells were subsequently infected with HIV-1(VSV-G) in the presence of dynasore for 2 hours. Following infection, cells were washed three times with medium, and then cultured in the absence of dynasore. Viral replication was monitored by flow <t>cytometry</t> analysis of HIV-dependent GFP expression at 48 hours (20,000 cells analyzed per sample). Propidum iodide (PI) was added into the cell suspension prior to flow cytometry. Viable cells were gated (R1) based on low PI staining and cell size (FSC). GFP expression within the viable cell population (R1) was measured. Both the GFP percentage (%) and mean intensity (M) were shown. (B) is an identical experiment using HIV-1 NL4-3 (Wt). For the 80 µM dynasore treatment, another three-independent infections with each virus were performed. The averages from the three-independent experiments are: 15.05%±0.21 (VSV-G), 10.11%±0.06 (VSV-G plus 80 µM dynasore), p = 0.0003; 2.94%±0.39 (Wt), 2.84%±0.30 (Wt plus 80 µM dynasore), p = 0.38.
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    Different effects of <t>dynasore</t> on the replication of HIV-1(VSV-G) and Wt in a human T cell, Rev-CEM. (A) Rev-CEM, a Rev-dependent GFP indicator cell, was pretreated for 30 minutes with 80 µM, 8 µM, or 0.8 µM dynasore, respectively, or treated with 0.1% DMSO as a control. Cells were subsequently infected with HIV-1(VSV-G) in the presence of dynasore for 2 hours. Following infection, cells were washed three times with medium, and then cultured in the absence of dynasore. Viral replication was monitored by flow <t>cytometry</t> analysis of HIV-dependent GFP expression at 48 hours (20,000 cells analyzed per sample). Propidum iodide (PI) was added into the cell suspension prior to flow cytometry. Viable cells were gated (R1) based on low PI staining and cell size (FSC). GFP expression within the viable cell population (R1) was measured. Both the GFP percentage (%) and mean intensity (M) were shown. (B) is an identical experiment using HIV-1 NL4-3 (Wt). For the 80 µM dynasore treatment, another three-independent infections with each virus were performed. The averages from the three-independent experiments are: 15.05%±0.21 (VSV-G), 10.11%±0.06 (VSV-G plus 80 µM dynasore), p = 0.0003; 2.94%±0.39 (Wt), 2.84%±0.30 (Wt plus 80 µM dynasore), p = 0.38.
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    Different effects of <t>dynasore</t> on the replication of HIV-1(VSV-G) and Wt in a human T cell, Rev-CEM. (A) Rev-CEM, a Rev-dependent GFP indicator cell, was pretreated for 30 minutes with 80 µM, 8 µM, or 0.8 µM dynasore, respectively, or treated with 0.1% DMSO as a control. Cells were subsequently infected with HIV-1(VSV-G) in the presence of dynasore for 2 hours. Following infection, cells were washed three times with medium, and then cultured in the absence of dynasore. Viral replication was monitored by flow <t>cytometry</t> analysis of HIV-dependent GFP expression at 48 hours (20,000 cells analyzed per sample). Propidum iodide (PI) was added into the cell suspension prior to flow cytometry. Viable cells were gated (R1) based on low PI staining and cell size (FSC). GFP expression within the viable cell population (R1) was measured. Both the GFP percentage (%) and mean intensity (M) were shown. (B) is an identical experiment using HIV-1 NL4-3 (Wt). For the 80 µM dynasore treatment, another three-independent infections with each virus were performed. The averages from the three-independent experiments are: 15.05%±0.21 (VSV-G), 10.11%±0.06 (VSV-G plus 80 µM dynasore), p = 0.0003; 2.94%±0.39 (Wt), 2.84%±0.30 (Wt plus 80 µM dynasore), p = 0.38.
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    Different effects of <t>dynasore</t> on the replication of HIV-1(VSV-G) and Wt in a human T cell, Rev-CEM. (A) Rev-CEM, a Rev-dependent GFP indicator cell, was pretreated for 30 minutes with 80 µM, 8 µM, or 0.8 µM dynasore, respectively, or treated with 0.1% DMSO as a control. Cells were subsequently infected with HIV-1(VSV-G) in the presence of dynasore for 2 hours. Following infection, cells were washed three times with medium, and then cultured in the absence of dynasore. Viral replication was monitored by flow <t>cytometry</t> analysis of HIV-dependent GFP expression at 48 hours (20,000 cells analyzed per sample). Propidum iodide (PI) was added into the cell suspension prior to flow cytometry. Viable cells were gated (R1) based on low PI staining and cell size (FSC). GFP expression within the viable cell population (R1) was measured. Both the GFP percentage (%) and mean intensity (M) were shown. (B) is an identical experiment using HIV-1 NL4-3 (Wt). For the 80 µM dynasore treatment, another three-independent infections with each virus were performed. The averages from the three-independent experiments are: 15.05%±0.21 (VSV-G), 10.11%±0.06 (VSV-G plus 80 µM dynasore), p = 0.0003; 2.94%±0.39 (Wt), 2.84%±0.30 (Wt plus 80 µM dynasore), p = 0.38.
    Dynamin, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 38 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Luciferase transfection of LPD (LP a D, LP b D, LP c D, and LP d D) and HLPD (HLP a D, HLP b D, HLP c D, and HLP d D) in SMMC-7721 cells incubated for 24 h. Notes: In the absence (normal group) and presence (dynasore group) of ( A ) anti-CD44 antibody (50 μg/mL) pretreated for 1 h, ( B ) <t>dynamin</t> inhibitor I with 0.2% DMSO in the solution (DI; dynasore, 80 μM) pretreated for 1 h, ( C ) chlorpromazine (CPZ; 20 μM) pretreated for 1 h, and ( D ) 5-( N -ethyl- N -isopropyl) amiloride (EIPA; 100 μM) with 0.1% DMSO in the solution pretreated for 30 min. Values are the means of four replicates ± SD. “*” Represents an alpha value of P
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    Luciferase transfection of LPD (LP a D, LP b D, LP c D, and LP d D) and HLPD (HLP a D, HLP b D, HLP c D, and HLP d D) in SMMC-7721 cells incubated for 24 h. Notes: In the absence (normal group) and presence (dynasore group) of ( A ) anti-CD44 antibody (50 μg/mL) pretreated for 1 h, ( B ) <t>dynamin</t> inhibitor I with 0.2% DMSO in the solution (DI; dynasore, 80 μM) pretreated for 1 h, ( C ) chlorpromazine (CPZ; 20 μM) pretreated for 1 h, and ( D ) 5-( N -ethyl- N -isopropyl) amiloride (EIPA; 100 μM) with 0.1% DMSO in the solution pretreated for 30 min. Values are the means of four replicates ± SD. “*” Represents an alpha value of P
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    Luciferase transfection of LPD (LP a D, LP b D, LP c D, and LP d D) and HLPD (HLP a D, HLP b D, HLP c D, and HLP d D) in SMMC-7721 cells incubated for 24 h. Notes: In the absence (normal group) and presence (dynasore group) of ( A ) anti-CD44 antibody (50 μg/mL) pretreated for 1 h, ( B ) <t>dynamin</t> inhibitor I with 0.2% DMSO in the solution (DI; dynasore, 80 μM) pretreated for 1 h, ( C ) chlorpromazine (CPZ; 20 μM) pretreated for 1 h, and ( D ) 5-( N -ethyl- N -isopropyl) amiloride (EIPA; 100 μM) with 0.1% DMSO in the solution pretreated for 30 min. Values are the means of four replicates ± SD. “*” Represents an alpha value of P
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    Luciferase transfection of LPD (LP a D, LP b D, LP c D, and LP d D) and HLPD (HLP a D, HLP b D, HLP c D, and HLP d D) in SMMC-7721 cells incubated for 24 h. Notes: In the absence (normal group) and presence (dynasore group) of ( A ) anti-CD44 antibody (50 μg/mL) pretreated for 1 h, ( B ) <t>dynamin</t> inhibitor I with 0.2% DMSO in the solution (DI; dynasore, 80 μM) pretreated for 1 h, ( C ) chlorpromazine (CPZ; 20 μM) pretreated for 1 h, and ( D ) 5-( N -ethyl- N -isopropyl) amiloride (EIPA; 100 μM) with 0.1% DMSO in the solution pretreated for 30 min. Values are the means of four replicates ± SD. “*” Represents an alpha value of P
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    Image Search Results


    Dynamin2 is essential for integrin-mediated static adhesion of human T cells. ( A - C ) Analysis of the adhesion of primary human resting CD4 + T cells to either ICAM-1-Fc or VCAM-1-Fc under static conditions. Lymphocytes were treated with DMSO as a control or 80μM dynasore to inhibit dynamin2 activity. If indicated, adhesion was stimulated with ( A , n = 3–5) 50ng/ml PMA, ( B , n = 3–5) 1μg/ml CXCL12 or ( C , n = 3) anti-CD3/CD28-coated beads (1:1 ratio to cells). 45min after seeding, the total number of adherent cells per mm 2 was quantified. ( D ) Detailed differential interference contrast (DIC) images of representative ICAM-1-Fc coated areas with adherent CD4 + T lymphocytes following treatment with DMSO or dynasore and, if indicated, stimulation with PMA or CXCL12. ( E , n = 4) Analysis of static adhesion of primary human resting CD4 + T cells to either ICAM-1-Fc or VCAM-1-Fc following treatment with dynole 31–2 as a control or dynole 34–2 to inhibit dynamin2 activity. If indicated, cells were stimulated with 50ng/ml PMA. 45min after seeding, the total number of adherent cells per mm 2 was quantified. ( F , n = 3) Analysis of static adhesion of primary human resting CD4 + T cells to either ICAM-1-Fc or VCAM-1-Fc 48h after the cells were transfected with either control siRNA or dynamin2 siRNA to knock down the large GTPase. If indicated, cells were stimulated with 50ng/ml PMA. 45min after seeding, the total number of adherent cells per mm 2 was quantified. Mean +SEM, *P≤0.05, **P≤0.01, ***P≤0.001.

    Journal: PLoS ONE

    Article Title: Dynamin2 controls Rap1 activation and integrin clustering in human T lymphocyte adhesion

    doi: 10.1371/journal.pone.0172443

    Figure Lengend Snippet: Dynamin2 is essential for integrin-mediated static adhesion of human T cells. ( A - C ) Analysis of the adhesion of primary human resting CD4 + T cells to either ICAM-1-Fc or VCAM-1-Fc under static conditions. Lymphocytes were treated with DMSO as a control or 80μM dynasore to inhibit dynamin2 activity. If indicated, adhesion was stimulated with ( A , n = 3–5) 50ng/ml PMA, ( B , n = 3–5) 1μg/ml CXCL12 or ( C , n = 3) anti-CD3/CD28-coated beads (1:1 ratio to cells). 45min after seeding, the total number of adherent cells per mm 2 was quantified. ( D ) Detailed differential interference contrast (DIC) images of representative ICAM-1-Fc coated areas with adherent CD4 + T lymphocytes following treatment with DMSO or dynasore and, if indicated, stimulation with PMA or CXCL12. ( E , n = 4) Analysis of static adhesion of primary human resting CD4 + T cells to either ICAM-1-Fc or VCAM-1-Fc following treatment with dynole 31–2 as a control or dynole 34–2 to inhibit dynamin2 activity. If indicated, cells were stimulated with 50ng/ml PMA. 45min after seeding, the total number of adherent cells per mm 2 was quantified. ( F , n = 3) Analysis of static adhesion of primary human resting CD4 + T cells to either ICAM-1-Fc or VCAM-1-Fc 48h after the cells were transfected with either control siRNA or dynamin2 siRNA to knock down the large GTPase. If indicated, cells were stimulated with 50ng/ml PMA. 45min after seeding, the total number of adherent cells per mm 2 was quantified. Mean +SEM, *P≤0.05, **P≤0.01, ***P≤0.001.

    Article Snippet: Chemical inhibitors The following small molecules were used for the chemical inhibition of specific proteins or cellular processes: brefeldin A (B6542, Sigma-Aldrich), chlorpromazine (C8138, Sigma-Aldrich), cytochalasin D (C8273, Sigma-Aldrich), dynasore hydrate (D7693, Sigma-Aldrich), dynole 34–2 (ab120463, Abcam), dynole 31–2 (ab120464, Abcam), Exo1 (#1850, Tocris), latrunculin A (L5163, Sigma-Aldrich), monodansylcadaverine (30432, Sigma-Aldrich).

    Techniques: Activity Assay, Transfection

    Adhesion-dependent T cell migration on 2D surfaces is regulated by dynamin2. ( A ) Migration tracks of primary human resting CD4 + T lymphocytes migrating on a 2D surface coated with ICAM-1-Fc for 30min. If indicated, migration was stimulated by adding 1μg/ml CXCL12 uniformly. Cells were incubated with either DMSO as a control or dynasore to inhibit dynamin2 activity. ( B ) Quantification of accumulated distance and ( C ) average migratory speed (velocity) of the migrating lymphocytes corresponding to ( A ). Results show one representative experiment out of three. ( D ) Quantification of chemotaxis of human resting CD4 + T cells through a polycarbonate transwell filter (3μm pore size) 4h after cells were seeded. If indicated, chemotaxis was stimulated by adding 200ng/ml CXCL12 to the lower well. Mean +SEM, n = 3. ( E ) Quantification of accumulated distance and ( F ) velocity of migrating human resting CD4 + T cells in a 3D collagen gel corresponding to ( G ). ( G ) Migration tracks of primary human resting CD4 + T lymphocytes migrating within a 3D collagen gel for 30min. If indicated, migration was stimulated by adding 1μg/ml CXCL12 uniformly. Results show one representative experiment out of three. ***P≤0.001, ns means not significant.

    Journal: PLoS ONE

    Article Title: Dynamin2 controls Rap1 activation and integrin clustering in human T lymphocyte adhesion

    doi: 10.1371/journal.pone.0172443

    Figure Lengend Snippet: Adhesion-dependent T cell migration on 2D surfaces is regulated by dynamin2. ( A ) Migration tracks of primary human resting CD4 + T lymphocytes migrating on a 2D surface coated with ICAM-1-Fc for 30min. If indicated, migration was stimulated by adding 1μg/ml CXCL12 uniformly. Cells were incubated with either DMSO as a control or dynasore to inhibit dynamin2 activity. ( B ) Quantification of accumulated distance and ( C ) average migratory speed (velocity) of the migrating lymphocytes corresponding to ( A ). Results show one representative experiment out of three. ( D ) Quantification of chemotaxis of human resting CD4 + T cells through a polycarbonate transwell filter (3μm pore size) 4h after cells were seeded. If indicated, chemotaxis was stimulated by adding 200ng/ml CXCL12 to the lower well. Mean +SEM, n = 3. ( E ) Quantification of accumulated distance and ( F ) velocity of migrating human resting CD4 + T cells in a 3D collagen gel corresponding to ( G ). ( G ) Migration tracks of primary human resting CD4 + T lymphocytes migrating within a 3D collagen gel for 30min. If indicated, migration was stimulated by adding 1μg/ml CXCL12 uniformly. Results show one representative experiment out of three. ***P≤0.001, ns means not significant.

    Article Snippet: Chemical inhibitors The following small molecules were used for the chemical inhibition of specific proteins or cellular processes: brefeldin A (B6542, Sigma-Aldrich), chlorpromazine (C8138, Sigma-Aldrich), cytochalasin D (C8273, Sigma-Aldrich), dynasore hydrate (D7693, Sigma-Aldrich), dynole 34–2 (ab120463, Abcam), dynole 31–2 (ab120464, Abcam), Exo1 (#1850, Tocris), latrunculin A (L5163, Sigma-Aldrich), monodansylcadaverine (30432, Sigma-Aldrich).

    Techniques: Migration, Incubation, Activity Assay, Chemotaxis Assay

    Chemical inhibition of dynamin2 does not affect integrin affinity regulation. ( A - F ) FACS analysis of the expression of different beta 2 -integrin (CD18) affinity states on the surface of primary human resting CD4 + T lymphocytes in suspension using specific monoclonal antibodies. Cells were either incubated with DMSO as a control or with dynasore to inhibit dynamin2 activity. If indicated, lymphocytes were stimulated with 50ng/ml PMA or 1mM Mn 2+ . Antibodies used were ( A , D , n = 6) KIM127, recognizing intermediate affinity CD18, ( B , E , n = 4) 327C and ( C , F , n = 6) mAb24, both recognizing high affinity CD18. One representative histogram ( A - C ) as well as the relative quantification of surface expression with the stimulated DMSO sample set to 1 ( D - F ) is depicted for each antibody. ( G , H ) Analysis of the static adhesion of primary human resting CD4 + T cells to an ICAM-1-Fc coated surface. If indicated, adhesion was stimulated with KIM185, an antibody mechanically inducing the high affinity conformation of CD18 by binding it on the cellular surface. ( G ) Detailed DIC-images showing representative ICAM-1-Fc coated areas with adherent CD4 + T lymphocytes following incubation with DMSO/dynasore. ( H , n = 4) Quantification of total adherent CD4 + T cells per mm 2 45min after seeding them on an ICAM-1-Fc coated surface. ( I ) Histogram depicting the results of a FACS analysis of the binding of KIM185 antibody to CD18 expressed on the surface of human resting CD4 + T cells following 2h of incubation with DMSO or dynasore. Mean +SEM, **P≤0.01, ***P≤0.001, ns means not significant.

    Journal: PLoS ONE

    Article Title: Dynamin2 controls Rap1 activation and integrin clustering in human T lymphocyte adhesion

    doi: 10.1371/journal.pone.0172443

    Figure Lengend Snippet: Chemical inhibition of dynamin2 does not affect integrin affinity regulation. ( A - F ) FACS analysis of the expression of different beta 2 -integrin (CD18) affinity states on the surface of primary human resting CD4 + T lymphocytes in suspension using specific monoclonal antibodies. Cells were either incubated with DMSO as a control or with dynasore to inhibit dynamin2 activity. If indicated, lymphocytes were stimulated with 50ng/ml PMA or 1mM Mn 2+ . Antibodies used were ( A , D , n = 6) KIM127, recognizing intermediate affinity CD18, ( B , E , n = 4) 327C and ( C , F , n = 6) mAb24, both recognizing high affinity CD18. One representative histogram ( A - C ) as well as the relative quantification of surface expression with the stimulated DMSO sample set to 1 ( D - F ) is depicted for each antibody. ( G , H ) Analysis of the static adhesion of primary human resting CD4 + T cells to an ICAM-1-Fc coated surface. If indicated, adhesion was stimulated with KIM185, an antibody mechanically inducing the high affinity conformation of CD18 by binding it on the cellular surface. ( G ) Detailed DIC-images showing representative ICAM-1-Fc coated areas with adherent CD4 + T lymphocytes following incubation with DMSO/dynasore. ( H , n = 4) Quantification of total adherent CD4 + T cells per mm 2 45min after seeding them on an ICAM-1-Fc coated surface. ( I ) Histogram depicting the results of a FACS analysis of the binding of KIM185 antibody to CD18 expressed on the surface of human resting CD4 + T cells following 2h of incubation with DMSO or dynasore. Mean +SEM, **P≤0.01, ***P≤0.001, ns means not significant.

    Article Snippet: Chemical inhibitors The following small molecules were used for the chemical inhibition of specific proteins or cellular processes: brefeldin A (B6542, Sigma-Aldrich), chlorpromazine (C8138, Sigma-Aldrich), cytochalasin D (C8273, Sigma-Aldrich), dynasore hydrate (D7693, Sigma-Aldrich), dynole 34–2 (ab120463, Abcam), dynole 31–2 (ab120464, Abcam), Exo1 (#1850, Tocris), latrunculin A (L5163, Sigma-Aldrich), monodansylcadaverine (30432, Sigma-Aldrich).

    Techniques: Inhibition, FACS, Expressing, Incubation, Activity Assay, Binding Assay

    Dynamin2 is essential for Rap1 activation, a crucial step in T lymphocyte adhesion. ( A - F ) Western blot analyses of biochemical pull-downs of activated GTP-bound Rap1 (and Ras-GTP in ( A )) via binding to immobilized GST-RalGDS-RBD (Rac1-GTP via GST-Pak1-PBD). Lysates were generated from primary human resting CD4 + T cells in suspension treated with ( A ) DMSO as a control or dynasore to inhibit dynamin2 activity, ( C ) dynole 31–2 as a control or dynole 34–2 to inhibit dynamin2 activity or ( E ) control siRNA or dynamin2 siRNA to knock down dynamin2 expression. If indicated, cells were stimulated by adding 50ng/ml PMA for 5min. ( B , D , F ) Quantifications of GTP-bound Rap1 corresponding to ( A ), ( C ) and ( E ), n = 3–4. ( G , H ) Analysis of primary human CD4 + T cells ( G ) or Jurkat E6.1 T cells ( H ) overexpressing either eGFP as a control, human Rap1a wildtype eGFP or a constitutively active Rap1a point mutant, Rap1a G12V eGFP. ( G , n = 2) The relative static adhesion to ICAM-1-Fc following treatment of the cells with DMSO or dynasore was examined. PMA-stimulated adhesion of DMSO-treated eGFP-overexpressing primary CD4 + T cells was set to one. ( H ) Activation of endogenously as well as overexpressed Rap1 was analyzed in Jurkat E6.1 T cells applying a biochemical pull-down via GST-Ral-GDS-RBD and western blotting. Mean +SEM, *P≤0.05, **P≤0.01.

    Journal: PLoS ONE

    Article Title: Dynamin2 controls Rap1 activation and integrin clustering in human T lymphocyte adhesion

    doi: 10.1371/journal.pone.0172443

    Figure Lengend Snippet: Dynamin2 is essential for Rap1 activation, a crucial step in T lymphocyte adhesion. ( A - F ) Western blot analyses of biochemical pull-downs of activated GTP-bound Rap1 (and Ras-GTP in ( A )) via binding to immobilized GST-RalGDS-RBD (Rac1-GTP via GST-Pak1-PBD). Lysates were generated from primary human resting CD4 + T cells in suspension treated with ( A ) DMSO as a control or dynasore to inhibit dynamin2 activity, ( C ) dynole 31–2 as a control or dynole 34–2 to inhibit dynamin2 activity or ( E ) control siRNA or dynamin2 siRNA to knock down dynamin2 expression. If indicated, cells were stimulated by adding 50ng/ml PMA for 5min. ( B , D , F ) Quantifications of GTP-bound Rap1 corresponding to ( A ), ( C ) and ( E ), n = 3–4. ( G , H ) Analysis of primary human CD4 + T cells ( G ) or Jurkat E6.1 T cells ( H ) overexpressing either eGFP as a control, human Rap1a wildtype eGFP or a constitutively active Rap1a point mutant, Rap1a G12V eGFP. ( G , n = 2) The relative static adhesion to ICAM-1-Fc following treatment of the cells with DMSO or dynasore was examined. PMA-stimulated adhesion of DMSO-treated eGFP-overexpressing primary CD4 + T cells was set to one. ( H ) Activation of endogenously as well as overexpressed Rap1 was analyzed in Jurkat E6.1 T cells applying a biochemical pull-down via GST-Ral-GDS-RBD and western blotting. Mean +SEM, *P≤0.05, **P≤0.01.

    Article Snippet: Chemical inhibitors The following small molecules were used for the chemical inhibition of specific proteins or cellular processes: brefeldin A (B6542, Sigma-Aldrich), chlorpromazine (C8138, Sigma-Aldrich), cytochalasin D (C8273, Sigma-Aldrich), dynasore hydrate (D7693, Sigma-Aldrich), dynole 34–2 (ab120463, Abcam), dynole 31–2 (ab120464, Abcam), Exo1 (#1850, Tocris), latrunculin A (L5163, Sigma-Aldrich), monodansylcadaverine (30432, Sigma-Aldrich).

    Techniques: Activation Assay, Western Blot, Binding Assay, Generated, Activity Assay, Expressing, Mutagenesis

    Integrin clustering strongly depends on dynamin2 in human CD4 + T cells. ( A , B ) Analysis of beta 2 -integrin clustering on primary human resting CD4 + T lymphocytes in suspension. Cells were incubated for 2h with either DMSO as a control or dynasore to inhibit dynamin2 activity. Subsequently, cells were fixed with paraformaldehyde and the beta 2 -integrin chain was immunofluorescently labeled using the monoclonal antibody MEM-48. Lymphocytes were seeded in microchannels and after settlement Z-stack images were acquired using a confocal laser scanning microscope. ( A , n = 4) T cells bearing prominent “high intensity” beta 2 -integrin clusters were quantified. ( B ) Representative maximum intensity projections of Z-stacks (0.3μm interval) depicting T lymphocytes in suspension following staining of beta 2 -integrin. ( C ) Analysis of beta 2 -integrin clustering on primary human resting CD4 + T lymphocytes exposed to coated surfaces. First, cells were covalently labeled with the cell tracer CFSE. Subsequently, they were incubated for 1h 45min with either DMSO as a control or dynasore to inhibit dynamin2 activity. Then beta 2 -integrins were stained using the monoclonal antibody MHM23. The lymphocytes were seeded for 45min on surfaces coated with either ICAM-1-Fc alone or a mixture of ICAM-1-Fc and anti-CD3/CD28 antibodies to mimic an antigen presenting cell. Finally, Z-stacks (0.3μm interval) were acquired using a confocal laser scanning microscope. Mean + SEM, **P≤0.01.

    Journal: PLoS ONE

    Article Title: Dynamin2 controls Rap1 activation and integrin clustering in human T lymphocyte adhesion

    doi: 10.1371/journal.pone.0172443

    Figure Lengend Snippet: Integrin clustering strongly depends on dynamin2 in human CD4 + T cells. ( A , B ) Analysis of beta 2 -integrin clustering on primary human resting CD4 + T lymphocytes in suspension. Cells were incubated for 2h with either DMSO as a control or dynasore to inhibit dynamin2 activity. Subsequently, cells were fixed with paraformaldehyde and the beta 2 -integrin chain was immunofluorescently labeled using the monoclonal antibody MEM-48. Lymphocytes were seeded in microchannels and after settlement Z-stack images were acquired using a confocal laser scanning microscope. ( A , n = 4) T cells bearing prominent “high intensity” beta 2 -integrin clusters were quantified. ( B ) Representative maximum intensity projections of Z-stacks (0.3μm interval) depicting T lymphocytes in suspension following staining of beta 2 -integrin. ( C ) Analysis of beta 2 -integrin clustering on primary human resting CD4 + T lymphocytes exposed to coated surfaces. First, cells were covalently labeled with the cell tracer CFSE. Subsequently, they were incubated for 1h 45min with either DMSO as a control or dynasore to inhibit dynamin2 activity. Then beta 2 -integrins were stained using the monoclonal antibody MHM23. The lymphocytes were seeded for 45min on surfaces coated with either ICAM-1-Fc alone or a mixture of ICAM-1-Fc and anti-CD3/CD28 antibodies to mimic an antigen presenting cell. Finally, Z-stacks (0.3μm interval) were acquired using a confocal laser scanning microscope. Mean + SEM, **P≤0.01.

    Article Snippet: Chemical inhibitors The following small molecules were used for the chemical inhibition of specific proteins or cellular processes: brefeldin A (B6542, Sigma-Aldrich), chlorpromazine (C8138, Sigma-Aldrich), cytochalasin D (C8273, Sigma-Aldrich), dynasore hydrate (D7693, Sigma-Aldrich), dynole 34–2 (ab120463, Abcam), dynole 31–2 (ab120464, Abcam), Exo1 (#1850, Tocris), latrunculin A (L5163, Sigma-Aldrich), monodansylcadaverine (30432, Sigma-Aldrich).

    Techniques: Incubation, Activity Assay, Labeling, Laser-Scanning Microscopy, Staining

    Dynamin2 regulates integrin-mediated T lymphocyte adhesion under laminar flow. Analysis of adhesion properties of primary human resting CD4 + T cells to a surface coated with ICAM-1-Fc and VCAM-1-Fc covered by CXCL12 under unidirectional laminar flow ( A - C 2.8 dyn/cm 2 , D - E 1 dyn/cm 2 ). ( A - C ) T Lymphocytes were either treated with DMSO as a control or with dynasore to inhibit dynamin2 activity. ( A ) Tracks of rolling and/or migrating T cells along the coated surface under unidirectional laminar flow. Points indicate adherent cells. ( B ) Phase contrast images showing representative areas of the coated surfaces with adherent T cells after 30min exposure to a cell suspension under laminar flow and subsequent washing with HBSS. ( C ) End point quantification of T lymphocyte adhesion under flow (corresponding to ( A ) and ( B ), n = 4). ( D ) End point quantification of T lymphocyte adhesion following treatment of the cells with either dynole 31–2 as a control or dynole 34–2 to inhibit dynamin2 activity (n = 6). ( E ) End point analysis of T cell adhesion after transfection of the lymphocytes with either control siRNA or siRNA oligos directed against dynamin2 (n = 2). Mean +SEM, ***P≤0.001.

    Journal: PLoS ONE

    Article Title: Dynamin2 controls Rap1 activation and integrin clustering in human T lymphocyte adhesion

    doi: 10.1371/journal.pone.0172443

    Figure Lengend Snippet: Dynamin2 regulates integrin-mediated T lymphocyte adhesion under laminar flow. Analysis of adhesion properties of primary human resting CD4 + T cells to a surface coated with ICAM-1-Fc and VCAM-1-Fc covered by CXCL12 under unidirectional laminar flow ( A - C 2.8 dyn/cm 2 , D - E 1 dyn/cm 2 ). ( A - C ) T Lymphocytes were either treated with DMSO as a control or with dynasore to inhibit dynamin2 activity. ( A ) Tracks of rolling and/or migrating T cells along the coated surface under unidirectional laminar flow. Points indicate adherent cells. ( B ) Phase contrast images showing representative areas of the coated surfaces with adherent T cells after 30min exposure to a cell suspension under laminar flow and subsequent washing with HBSS. ( C ) End point quantification of T lymphocyte adhesion under flow (corresponding to ( A ) and ( B ), n = 4). ( D ) End point quantification of T lymphocyte adhesion following treatment of the cells with either dynole 31–2 as a control or dynole 34–2 to inhibit dynamin2 activity (n = 6). ( E ) End point analysis of T cell adhesion after transfection of the lymphocytes with either control siRNA or siRNA oligos directed against dynamin2 (n = 2). Mean +SEM, ***P≤0.001.

    Article Snippet: Chemical inhibitors The following small molecules were used for the chemical inhibition of specific proteins or cellular processes: brefeldin A (B6542, Sigma-Aldrich), chlorpromazine (C8138, Sigma-Aldrich), cytochalasin D (C8273, Sigma-Aldrich), dynasore hydrate (D7693, Sigma-Aldrich), dynole 34–2 (ab120463, Abcam), dynole 31–2 (ab120464, Abcam), Exo1 (#1850, Tocris), latrunculin A (L5163, Sigma-Aldrich), monodansylcadaverine (30432, Sigma-Aldrich).

    Techniques: Flow Cytometry, Activity Assay, Transfection

    Dynamin2 eGFP colocalizes with and regulates the activity of different signaling molecules at the basal plasma membrane. ( A , C ) Dynamin2 eGFP was overexpressed in Jurkat E6.1 T cells. Cells were seeded on a surface coated with ICAM-1-Fc and activating anti-CD3 (10μg/ml) and anti-CD28 (20μg/ml) antibodies for 15min. After fixation, F-actin was labeled using TRITC-phalloidin and immunofluorescence staining of the following proteins was performed: phosphorylated focal adhesion kinase (FAK, Tyr397), phosphorylated protein tyrosine kinase 2 (Pyk2, Tyr402), phosphorylated SRC familiy kinases (SFKs, Tyr416 or equivalent), CD18, talin1. Images were acquired using a confocal laser scanning microscope with focus on the basal plasma membrane. ( B ) Western blot analysis of Jurkat E6.1 T cells or primary human CD4 + T cells which were either treated with dynasore to inhibit dynamin2 activity or with DMSO as a solvent control. Cells were seeded on either uncoated, ICAM-1-Fc-coated or ICAM-1-Fc/anti-CD3/anti-CD28-coated surfaces for 15min. Phosphorylation of FAK and Pyk2 were analyzed.

    Journal: PLoS ONE

    Article Title: Dynamin2 controls Rap1 activation and integrin clustering in human T lymphocyte adhesion

    doi: 10.1371/journal.pone.0172443

    Figure Lengend Snippet: Dynamin2 eGFP colocalizes with and regulates the activity of different signaling molecules at the basal plasma membrane. ( A , C ) Dynamin2 eGFP was overexpressed in Jurkat E6.1 T cells. Cells were seeded on a surface coated with ICAM-1-Fc and activating anti-CD3 (10μg/ml) and anti-CD28 (20μg/ml) antibodies for 15min. After fixation, F-actin was labeled using TRITC-phalloidin and immunofluorescence staining of the following proteins was performed: phosphorylated focal adhesion kinase (FAK, Tyr397), phosphorylated protein tyrosine kinase 2 (Pyk2, Tyr402), phosphorylated SRC familiy kinases (SFKs, Tyr416 or equivalent), CD18, talin1. Images were acquired using a confocal laser scanning microscope with focus on the basal plasma membrane. ( B ) Western blot analysis of Jurkat E6.1 T cells or primary human CD4 + T cells which were either treated with dynasore to inhibit dynamin2 activity or with DMSO as a solvent control. Cells were seeded on either uncoated, ICAM-1-Fc-coated or ICAM-1-Fc/anti-CD3/anti-CD28-coated surfaces for 15min. Phosphorylation of FAK and Pyk2 were analyzed.

    Article Snippet: Chemical inhibitors The following small molecules were used for the chemical inhibition of specific proteins or cellular processes: brefeldin A (B6542, Sigma-Aldrich), chlorpromazine (C8138, Sigma-Aldrich), cytochalasin D (C8273, Sigma-Aldrich), dynasore hydrate (D7693, Sigma-Aldrich), dynole 34–2 (ab120463, Abcam), dynole 31–2 (ab120464, Abcam), Exo1 (#1850, Tocris), latrunculin A (L5163, Sigma-Aldrich), monodansylcadaverine (30432, Sigma-Aldrich).

    Techniques: Activity Assay, Labeling, Immunofluorescence, Staining, Laser-Scanning Microscopy, Western Blot

    There is no evidence for a direct interaction of dynamin2 and Rap1 but dynamin2 colocalizes with phosphorylated RapGEF1 and regulates its activity. ( A-B ) Western blot analyses of immunoprecipitations using either anti-dynamin2 ( A ) or anti-RapGEF1 ( B ) antibodies and their respective isotype controls coupled to Protein G-coated dynabeads. Resting human primary CD4 + T cells were stimulated with 50ng/ml PMA if indicated. ( C-D ) Dynamin2 eGFP (in ( C ) Rap1a RFP as well) was overexpressed in Jurkat E6.1 T cells. Cells were seeded on a surface coated with ICAM-1-Fc and activating anti-CD3 (10μg/ml) and anti-CD28 (20μg/ml) antibodies for 15min. After fixation, immunofluorescence staining of phosphorylated RapGEF1 (Tyr504) was performed ( D ). Images were acquired using a confocal laser scanning microscope with focus on the basal plasma membrane. ( E ) Western blot analysis of primary human CD4 + T cells either treated with dynasore to inhibit dynamin2 activity or with DMSO as a solvent control. Cells were seeded on either uncoated, ICAM-1-Fc-coated or ICAM-1-Fc/anti-CD3/anti-CD28-coated surfaces for 15min. Phosphorylation status of RapGEF1 was analyzed. ( F ) Quantification of ( E ) with n = 4. Mean +SEM, ***P≤0.001.

    Journal: PLoS ONE

    Article Title: Dynamin2 controls Rap1 activation and integrin clustering in human T lymphocyte adhesion

    doi: 10.1371/journal.pone.0172443

    Figure Lengend Snippet: There is no evidence for a direct interaction of dynamin2 and Rap1 but dynamin2 colocalizes with phosphorylated RapGEF1 and regulates its activity. ( A-B ) Western blot analyses of immunoprecipitations using either anti-dynamin2 ( A ) or anti-RapGEF1 ( B ) antibodies and their respective isotype controls coupled to Protein G-coated dynabeads. Resting human primary CD4 + T cells were stimulated with 50ng/ml PMA if indicated. ( C-D ) Dynamin2 eGFP (in ( C ) Rap1a RFP as well) was overexpressed in Jurkat E6.1 T cells. Cells were seeded on a surface coated with ICAM-1-Fc and activating anti-CD3 (10μg/ml) and anti-CD28 (20μg/ml) antibodies for 15min. After fixation, immunofluorescence staining of phosphorylated RapGEF1 (Tyr504) was performed ( D ). Images were acquired using a confocal laser scanning microscope with focus on the basal plasma membrane. ( E ) Western blot analysis of primary human CD4 + T cells either treated with dynasore to inhibit dynamin2 activity or with DMSO as a solvent control. Cells were seeded on either uncoated, ICAM-1-Fc-coated or ICAM-1-Fc/anti-CD3/anti-CD28-coated surfaces for 15min. Phosphorylation status of RapGEF1 was analyzed. ( F ) Quantification of ( E ) with n = 4. Mean +SEM, ***P≤0.001.

    Article Snippet: Chemical inhibitors The following small molecules were used for the chemical inhibition of specific proteins or cellular processes: brefeldin A (B6542, Sigma-Aldrich), chlorpromazine (C8138, Sigma-Aldrich), cytochalasin D (C8273, Sigma-Aldrich), dynasore hydrate (D7693, Sigma-Aldrich), dynole 34–2 (ab120463, Abcam), dynole 31–2 (ab120464, Abcam), Exo1 (#1850, Tocris), latrunculin A (L5163, Sigma-Aldrich), monodansylcadaverine (30432, Sigma-Aldrich).

    Techniques: Activity Assay, Western Blot, Immunofluorescence, Staining, Laser-Scanning Microscopy

    Dynamin2 partially regulates F-actin polymerization, which is dispensable for static adhesion of human resting CD4 + T cells. ( A ) Maximum intensity projections of confocal images from Z-stacks (0.3μm interval) of phalloidin-TRITC labeled resting human CD4 + T cells in suspension. If indicated, F-actin polymerization and/or cell polarization were stimulated by adding 1μg/ml CXCL12 or 50ng/ml PMA. ( B , n = 5) FACS analysis of F-actin expression in primary resting human CD4 + T cells in suspension. Lymphocytes were either treated with DMSO as a control or with dynasore to inhibit dynamin2 activity. If indicated, actin polymerization was stimulated by adding 50ng/ml PMA for 45min. F-actin was specifically labeled by adding phalloidin-TRITC. ( C , D ) Analysis of static adhesion of primary human resting CD4 + T cells to either ICAM-1-Fc or VCAM-1-Fc following treatment of the cells with different F-actin polymerization inhibitors. If indicated, cells were stimulated with 50ng/ml PMA. 45min after seeding, the total number of adherent cells per mm 2 was quantified. F-actin polymerization was inhibited with ( C , n = 4) cytochalasin D or ( D , n = 3) latrunculin A. ( E ) Detailed DIC images of representative ICAM-1-Fc or VCAM-1-Fc coated areas with adherent CD4 + T lymphocytes following treatment with F-actin polymerization inhibitors and stimulation with PMA. Mean +SEM, **P≤0.01, ns means not significant.

    Journal: PLoS ONE

    Article Title: Dynamin2 controls Rap1 activation and integrin clustering in human T lymphocyte adhesion

    doi: 10.1371/journal.pone.0172443

    Figure Lengend Snippet: Dynamin2 partially regulates F-actin polymerization, which is dispensable for static adhesion of human resting CD4 + T cells. ( A ) Maximum intensity projections of confocal images from Z-stacks (0.3μm interval) of phalloidin-TRITC labeled resting human CD4 + T cells in suspension. If indicated, F-actin polymerization and/or cell polarization were stimulated by adding 1μg/ml CXCL12 or 50ng/ml PMA. ( B , n = 5) FACS analysis of F-actin expression in primary resting human CD4 + T cells in suspension. Lymphocytes were either treated with DMSO as a control or with dynasore to inhibit dynamin2 activity. If indicated, actin polymerization was stimulated by adding 50ng/ml PMA for 45min. F-actin was specifically labeled by adding phalloidin-TRITC. ( C , D ) Analysis of static adhesion of primary human resting CD4 + T cells to either ICAM-1-Fc or VCAM-1-Fc following treatment of the cells with different F-actin polymerization inhibitors. If indicated, cells were stimulated with 50ng/ml PMA. 45min after seeding, the total number of adherent cells per mm 2 was quantified. F-actin polymerization was inhibited with ( C , n = 4) cytochalasin D or ( D , n = 3) latrunculin A. ( E ) Detailed DIC images of representative ICAM-1-Fc or VCAM-1-Fc coated areas with adherent CD4 + T lymphocytes following treatment with F-actin polymerization inhibitors and stimulation with PMA. Mean +SEM, **P≤0.01, ns means not significant.

    Article Snippet: Chemical inhibitors The following small molecules were used for the chemical inhibition of specific proteins or cellular processes: brefeldin A (B6542, Sigma-Aldrich), chlorpromazine (C8138, Sigma-Aldrich), cytochalasin D (C8273, Sigma-Aldrich), dynasore hydrate (D7693, Sigma-Aldrich), dynole 34–2 (ab120463, Abcam), dynole 31–2 (ab120464, Abcam), Exo1 (#1850, Tocris), latrunculin A (L5163, Sigma-Aldrich), monodansylcadaverine (30432, Sigma-Aldrich).

    Techniques: Labeling, FACS, Expressing, Activity Assay

    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,

    Journal: Journal of Virology

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

    doi: 10.1128/JVI.00331-10

    Figure Lengend 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,

    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.

    Techniques: Infection, Concentration Assay

    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

    Journal: Nature Communications

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

    doi: 10.1038/ncomms14508

    Figure Lengend 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

    Article Snippet: Reagents and antibodies The following reagents were used: mouse Sema3B-Fc (10 nM, R & D Systems), mouse Sema3C-Fc (10 nM, R & D Systems), mouse Sema3E-Fc (10 nM, R & D Systems), Texas Red-X phalloidin (1:50, Life Technologies), calcein-AM (1 μM, Sigma-Aldrich), Pitstop 2 and Pitstop 2-negative control (10 μM, Abcam) and dynasore (80 μM, Sigma-Aldrich).

    Techniques: 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

    Journal: Nature Communications

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

    doi: 10.1038/ncomms14508

    Figure Lengend 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

    Article Snippet: Reagents and antibodies The following reagents were used: mouse Sema3B-Fc (10 nM, R & D Systems), mouse Sema3C-Fc (10 nM, R & D Systems), mouse Sema3E-Fc (10 nM, R & D Systems), Texas Red-X phalloidin (1:50, Life Technologies), calcein-AM (1 μM, Sigma-Aldrich), Pitstop 2 and Pitstop 2-negative control (10 μM, Abcam) and dynasore (80 μM, Sigma-Aldrich).

    Techniques: Staining, Cell Culture, Expressing

    Inhibition of endocytic trafficking attenuates number and acidification of endolysosomes to halt autophagy flux. (a and b) Inhibition of endocytic trafficking in fibroblasts, with either dynasore or ciliobrevin A, consequently reduces the number of acidic vesicles indicated by LysoTracker Red fluorescence staining. Scale bar: 10 μm. Bar graphs in (b) represent the quantitative analysis of the LysoTracker Red fluorescence intensity and an average number of puncta per cell in both conditions. (c) Autophagy flux assay of HEK293T cells treated with DMSO and starved of amino acids for the indicated time points. Normal autophagy flux is indicated by the gradual decrease in the levels of LC3-II and SQSTM1 during starvation and their accumulation when the lysosomal function is inhibited by chloroquine. Bar graphs represent a quantitative analysis of the relative SQSTM1 and LC3 protein levels during starvation. (d) Autophagy flux was impaired in cells treated with either dynasore or ciliobrevin A. HEK293T Cells were pre-incubated with dynasore for 4 h or ciliobrevin A for 2 h prior to amino acid starvation for the indicated time points. Compared to controls (c), LC3-II protein levels did not decrease, but rather increased over time indicating an impaired autophagy flux due to lysosomal inhibition. Bar graphs represent a quantitative analysis of the relative SQSTM1 and LC3 protein levels during starvation. (e) Autophagic markers LC3 and SQSTM1 were decreased in cells overexpressing Flag-TFEB in an endocytic trafficking-dependent manner. Inhibition of dynein-dependent trafficking with ciliobrevin A strongly increased LC3-II and SQSTM1 protein levels. Bar graphs represent a quantitative analysis of the relative SQSTM1, LC3 and Flag-TFEB protein levels during starvation. A significant change relative to the control at the time point 0 h is given. Treatment with dynasore or ciliobrevin A did not affect fusion of autophagosomes with lysosomes (Figure S4). Data are represented as mean ± SEM, n = 6; ns denotes no significant difference, *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ANOVA.

    Journal: Autophagy

    Article Title: TFEB-driven endocytosis coordinates MTORC1 signaling and autophagy

    doi: 10.1080/15548627.2018.1511504

    Figure Lengend Snippet: Inhibition of endocytic trafficking attenuates number and acidification of endolysosomes to halt autophagy flux. (a and b) Inhibition of endocytic trafficking in fibroblasts, with either dynasore or ciliobrevin A, consequently reduces the number of acidic vesicles indicated by LysoTracker Red fluorescence staining. Scale bar: 10 μm. Bar graphs in (b) represent the quantitative analysis of the LysoTracker Red fluorescence intensity and an average number of puncta per cell in both conditions. (c) Autophagy flux assay of HEK293T cells treated with DMSO and starved of amino acids for the indicated time points. Normal autophagy flux is indicated by the gradual decrease in the levels of LC3-II and SQSTM1 during starvation and their accumulation when the lysosomal function is inhibited by chloroquine. Bar graphs represent a quantitative analysis of the relative SQSTM1 and LC3 protein levels during starvation. (d) Autophagy flux was impaired in cells treated with either dynasore or ciliobrevin A. HEK293T Cells were pre-incubated with dynasore for 4 h or ciliobrevin A for 2 h prior to amino acid starvation for the indicated time points. Compared to controls (c), LC3-II protein levels did not decrease, but rather increased over time indicating an impaired autophagy flux due to lysosomal inhibition. Bar graphs represent a quantitative analysis of the relative SQSTM1 and LC3 protein levels during starvation. (e) Autophagic markers LC3 and SQSTM1 were decreased in cells overexpressing Flag-TFEB in an endocytic trafficking-dependent manner. Inhibition of dynein-dependent trafficking with ciliobrevin A strongly increased LC3-II and SQSTM1 protein levels. Bar graphs represent a quantitative analysis of the relative SQSTM1, LC3 and Flag-TFEB protein levels during starvation. A significant change relative to the control at the time point 0 h is given. Treatment with dynasore or ciliobrevin A did not affect fusion of autophagosomes with lysosomes (Figure S4). Data are represented as mean ± SEM, n = 6; ns denotes no significant difference, *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ANOVA.

    Article Snippet: Other materials used for cell culture and culture treatments include HBSS 10X (Life Technologies, 14,065–056), hydroxyl-dynasore (Sigma-Aldrich, SML0340), ciliobrevin A (Sigma-Aldrich, 4529), Dulbecco’s modified Eagle’s medium (Sigma-Aldrich, D5671), fetal bovine serum (FBS; Life Technologies, 10,438), dialyzed FBS (dFBS; Life Technologies, 26,400,044), chloroquine (Sigma-Aldrich, C6628), Torin 1 (Tocris, 4247), and BioT transfection reagent (Bioland Scientific, B01-00).

    Techniques: Inhibition, Fluorescence, Staining, Flux Assay, Incubation

    Endocytic trafficking controls MTORC1 tethering and signaling. (a-c) Pharmacological inhibition of DNM GTPase with dynasore under fed conditions disperses MTORC1 into the cytosol while TSC2, the negative regulator of MTORC1, relocalizes to lysosomes. NIH-3T3 fibroblasts were treated with 80 μM of dynasore for 4 h followed by colocalization analyses of MTOR or TSC2 with the lysosomal LAMP2 protein. The bar graph in (c) represents a quantitative evaluation of lysosomal MTORC1 or TSC2 shown in (a and b). Scale bars: 10 μm. (d) Dynasore treatment inhibits MTORC1 activity under fed conditions and has no significant effect on relative p-AKT (Thr308) protein levels. NIH-3T3 fibroblasts were subjected to hydroxyl-dynasore treatment for the indicated time points followed by p-RPS6KB1 immunoblot analyses for MTORC1 activity. Bar graph represents a quantitative analysis of relative protein levels of p-RPS6KB1 and p-AKT (Thr308). (e) Competitive inhibition of DNM2 with a dominant-negative form of DNM2 (DNM2 K44A ) reduced MTORC1 activity, whereas overexpression of the wild-type form increased MTORC1 signaling in HEK293T cells. An increasing LC3-II level, indicative of autophagy induction and buildup of autophagosomes, were observed in DNM2 K44A -overexpressing cells. Bar graph represents the quantitative analyses of relative protein levels of p-RPS6KB1, LC3-I and LC3-II. (f) Pharmacological inhibition of the dynein motor protein, hence retrograde trafficking, with ciliobrevin A dissociates MTOR from lysosomal membranes. NIH-3T3 fibroblasts were treated with 100 μM of ciliobrevin A for 2 h followed by colocalization analyses of MTOR with lysosomal LAMP2 protein. Scale bar: 10 μm. Bar graph represents quantitative analyses of colocalization of MTOR and LAMP2 signals. (g) Competitive inhibition of RAB5 with a dominant-negative form of RAB5 N133I reduced MTORC1 activity whereas the wild-type form increased MTORC1 signaling in HEK293T cells. Bar graph represents the quantitative analyses of relative protein levels of p-RPS6KB1. Inhibition of cellular endocytosis with hypertonic sucrose media and pharmacological inhibition of dynein motor proteins also inhibited MTORC1 signaling (see Figure S3). Data are represented as mean ± SEM, n = 3, p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ANOVA.

    Journal: Autophagy

    Article Title: TFEB-driven endocytosis coordinates MTORC1 signaling and autophagy

    doi: 10.1080/15548627.2018.1511504

    Figure Lengend Snippet: Endocytic trafficking controls MTORC1 tethering and signaling. (a-c) Pharmacological inhibition of DNM GTPase with dynasore under fed conditions disperses MTORC1 into the cytosol while TSC2, the negative regulator of MTORC1, relocalizes to lysosomes. NIH-3T3 fibroblasts were treated with 80 μM of dynasore for 4 h followed by colocalization analyses of MTOR or TSC2 with the lysosomal LAMP2 protein. The bar graph in (c) represents a quantitative evaluation of lysosomal MTORC1 or TSC2 shown in (a and b). Scale bars: 10 μm. (d) Dynasore treatment inhibits MTORC1 activity under fed conditions and has no significant effect on relative p-AKT (Thr308) protein levels. NIH-3T3 fibroblasts were subjected to hydroxyl-dynasore treatment for the indicated time points followed by p-RPS6KB1 immunoblot analyses for MTORC1 activity. Bar graph represents a quantitative analysis of relative protein levels of p-RPS6KB1 and p-AKT (Thr308). (e) Competitive inhibition of DNM2 with a dominant-negative form of DNM2 (DNM2 K44A ) reduced MTORC1 activity, whereas overexpression of the wild-type form increased MTORC1 signaling in HEK293T cells. An increasing LC3-II level, indicative of autophagy induction and buildup of autophagosomes, were observed in DNM2 K44A -overexpressing cells. Bar graph represents the quantitative analyses of relative protein levels of p-RPS6KB1, LC3-I and LC3-II. (f) Pharmacological inhibition of the dynein motor protein, hence retrograde trafficking, with ciliobrevin A dissociates MTOR from lysosomal membranes. NIH-3T3 fibroblasts were treated with 100 μM of ciliobrevin A for 2 h followed by colocalization analyses of MTOR with lysosomal LAMP2 protein. Scale bar: 10 μm. Bar graph represents quantitative analyses of colocalization of MTOR and LAMP2 signals. (g) Competitive inhibition of RAB5 with a dominant-negative form of RAB5 N133I reduced MTORC1 activity whereas the wild-type form increased MTORC1 signaling in HEK293T cells. Bar graph represents the quantitative analyses of relative protein levels of p-RPS6KB1. Inhibition of cellular endocytosis with hypertonic sucrose media and pharmacological inhibition of dynein motor proteins also inhibited MTORC1 signaling (see Figure S3). Data are represented as mean ± SEM, n = 3, p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ANOVA.

    Article Snippet: Other materials used for cell culture and culture treatments include HBSS 10X (Life Technologies, 14,065–056), hydroxyl-dynasore (Sigma-Aldrich, SML0340), ciliobrevin A (Sigma-Aldrich, 4529), Dulbecco’s modified Eagle’s medium (Sigma-Aldrich, D5671), fetal bovine serum (FBS; Life Technologies, 10,438), dialyzed FBS (dFBS; Life Technologies, 26,400,044), chloroquine (Sigma-Aldrich, C6628), Torin 1 (Tocris, 4247), and BioT transfection reagent (Bioland Scientific, B01-00).

    Techniques: Inhibition, Activity Assay, Dominant Negative Mutation, Over Expression

    Recovery of MTORC1 activity during prolonged starvation occurs in a TFEB-driven endocytosis-dependent manner. (a) MTORC1 activity was assessed in amino acid (leucine and glutamine)-starved HEK293T cells for the indicated time points. Relative p-RPS6KB1 (p-Thr389) as well as relative p-ULK1 (p-Ser757) levels were assessed by immunoblots indicative of changes in MTORC1 activity. Reactivation of MTORC1 was observed after 6 h of starvation in DMSO-treated control cells but remained suppressed in dynasore- or ciliobrevin A-treated and starved cells. Bar graphs represent a quantitative evaluation of relative p-RPS6KB1 and p-ULK1 protein levels. (b) Faster recovery of MTORC1 signaling was observed in HEK293T cells overexpressing WT DNM2, whereas the overexpression of the dominant negative form of DNM2 efficiently inhibited MTORC1 reactivation during starvation (compare DNM2 WT - and DNM2 K44A -expressing cells to those expressing an empty vector control). Arrows indicate specific p-RPS6KB1 bands. Bar graphs represent the quantitative analyses of relative p-RPS6KB1 protein levels. (c and d) Depletion of endogenous TFEB inhibits MTORC1 reactivation, whereas overexpression of Flag-TFEB increased basal MTORC1 activity and promoted faster recovery of MTORC1 signaling during prolonged starvation in a DNM-dependent manner. An empty vector control is shown in the first lane and used for comparison to TFEB knockdown or overexpression samples. Bar graphs represent a quantitative evaluation of relative p-RPS6KB1 and p-ULK1 protein levels. Gray bars in (d) show relative p-RPS6KB1 levels adjusted for transfection efficiency of the Flag-TFEB vectors. Data are represented as mean ± SEM, n = 3, ns denotes ‘no significant difference’, *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ANOVA.

    Journal: Autophagy

    Article Title: TFEB-driven endocytosis coordinates MTORC1 signaling and autophagy

    doi: 10.1080/15548627.2018.1511504

    Figure Lengend Snippet: Recovery of MTORC1 activity during prolonged starvation occurs in a TFEB-driven endocytosis-dependent manner. (a) MTORC1 activity was assessed in amino acid (leucine and glutamine)-starved HEK293T cells for the indicated time points. Relative p-RPS6KB1 (p-Thr389) as well as relative p-ULK1 (p-Ser757) levels were assessed by immunoblots indicative of changes in MTORC1 activity. Reactivation of MTORC1 was observed after 6 h of starvation in DMSO-treated control cells but remained suppressed in dynasore- or ciliobrevin A-treated and starved cells. Bar graphs represent a quantitative evaluation of relative p-RPS6KB1 and p-ULK1 protein levels. (b) Faster recovery of MTORC1 signaling was observed in HEK293T cells overexpressing WT DNM2, whereas the overexpression of the dominant negative form of DNM2 efficiently inhibited MTORC1 reactivation during starvation (compare DNM2 WT - and DNM2 K44A -expressing cells to those expressing an empty vector control). Arrows indicate specific p-RPS6KB1 bands. Bar graphs represent the quantitative analyses of relative p-RPS6KB1 protein levels. (c and d) Depletion of endogenous TFEB inhibits MTORC1 reactivation, whereas overexpression of Flag-TFEB increased basal MTORC1 activity and promoted faster recovery of MTORC1 signaling during prolonged starvation in a DNM-dependent manner. An empty vector control is shown in the first lane and used for comparison to TFEB knockdown or overexpression samples. Bar graphs represent a quantitative evaluation of relative p-RPS6KB1 and p-ULK1 protein levels. Gray bars in (d) show relative p-RPS6KB1 levels adjusted for transfection efficiency of the Flag-TFEB vectors. Data are represented as mean ± SEM, n = 3, ns denotes ‘no significant difference’, *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ANOVA.

    Article Snippet: Other materials used for cell culture and culture treatments include HBSS 10X (Life Technologies, 14,065–056), hydroxyl-dynasore (Sigma-Aldrich, SML0340), ciliobrevin A (Sigma-Aldrich, 4529), Dulbecco’s modified Eagle’s medium (Sigma-Aldrich, D5671), fetal bovine serum (FBS; Life Technologies, 10,438), dialyzed FBS (dFBS; Life Technologies, 26,400,044), chloroquine (Sigma-Aldrich, C6628), Torin 1 (Tocris, 4247), and BioT transfection reagent (Bioland Scientific, B01-00).

    Techniques: Activity Assay, Western Blot, Over Expression, Dominant Negative Mutation, Expressing, Plasmid Preparation, Transfection

    Inhibition of endosomal trafficking translocates TFEB into the nucleus and reduces the quantity of LAMP2-positive vesicles. (a-c) Inhibition of DNM or dynein with dynasore or ciliobrevin A relocalizes TFEB to the nucleus. A plasmid expressing GFP-TFEB was transiently transfected into HeLa cells followed by dynasore (a and b) or ciliobrevin A treatment (A and C) with the indicated concentrations overnight. Nuclear TFEB was assessed by confocal microscopy and quantitatively represented in the graphs in (b and c). Scale bar: 10 μm. (D) Quantitative RT-PCR analyses of ciliobrevin A- and dynasore-treated cells showed increased transcription of TFEB target genes ( LC3, SQSTM1, MCOLN1, CTSB, CTSF , and TFEB ) after inhibition of endocytosis with dynasore or inhibition of retrograde trafficking with ciliobrevin A. Bar graphs represent fold change in mRNA levels. (e and f) NIH3T3 cells treated with dynasore or ciliobrevin A overnight showed a strong reduction of the intensity of LAMP2-immunoreactivity and number of LAMP2-positive spots per cell, indicative of the number of lysosomes. Right panels are magnifications of outlined areas. Bar graphs represent the quantitative analysis of the LAMP2 signal intensity and an average number of LAMP2 puncta per cell in both conditions. Scale bar: 10 μm. Data are represented as mean ± SEM, n = 3; *p ≤ 0.05; **p ≤ 0.01; ANOVA.

    Journal: Autophagy

    Article Title: TFEB-driven endocytosis coordinates MTORC1 signaling and autophagy

    doi: 10.1080/15548627.2018.1511504

    Figure Lengend Snippet: Inhibition of endosomal trafficking translocates TFEB into the nucleus and reduces the quantity of LAMP2-positive vesicles. (a-c) Inhibition of DNM or dynein with dynasore or ciliobrevin A relocalizes TFEB to the nucleus. A plasmid expressing GFP-TFEB was transiently transfected into HeLa cells followed by dynasore (a and b) or ciliobrevin A treatment (A and C) with the indicated concentrations overnight. Nuclear TFEB was assessed by confocal microscopy and quantitatively represented in the graphs in (b and c). Scale bar: 10 μm. (D) Quantitative RT-PCR analyses of ciliobrevin A- and dynasore-treated cells showed increased transcription of TFEB target genes ( LC3, SQSTM1, MCOLN1, CTSB, CTSF , and TFEB ) after inhibition of endocytosis with dynasore or inhibition of retrograde trafficking with ciliobrevin A. Bar graphs represent fold change in mRNA levels. (e and f) NIH3T3 cells treated with dynasore or ciliobrevin A overnight showed a strong reduction of the intensity of LAMP2-immunoreactivity and number of LAMP2-positive spots per cell, indicative of the number of lysosomes. Right panels are magnifications of outlined areas. Bar graphs represent the quantitative analysis of the LAMP2 signal intensity and an average number of LAMP2 puncta per cell in both conditions. Scale bar: 10 μm. Data are represented as mean ± SEM, n = 3; *p ≤ 0.05; **p ≤ 0.01; ANOVA.

    Article Snippet: Other materials used for cell culture and culture treatments include HBSS 10X (Life Technologies, 14,065–056), hydroxyl-dynasore (Sigma-Aldrich, SML0340), ciliobrevin A (Sigma-Aldrich, 4529), Dulbecco’s modified Eagle’s medium (Sigma-Aldrich, D5671), fetal bovine serum (FBS; Life Technologies, 10,438), dialyzed FBS (dFBS; Life Technologies, 26,400,044), chloroquine (Sigma-Aldrich, C6628), Torin 1 (Tocris, 4247), and BioT transfection reagent (Bioland Scientific, B01-00).

    Techniques: Inhibition, Plasmid Preparation, Expressing, Transfection, Confocal Microscopy, Quantitative RT-PCR

    Morphological changes upon combined Dynasore-E2 treatment of MCF-7 cells. Semithin frozen sections of control MCF-7 cells labeled with antibodies directed against ER-α (green) and caveolin-1 (red). a – c ER-α receptor labeling occurred inside the cytoplasm and the plasma membrane in MCF-7 cells. Nuclear occurrence of the receptor could also be observed, however, to a lesser extent. The merged image of ER-α and caveolin-1 double labeling shows overlapping areas at the plasma membrane (arrowheads), while intracytoplasmic co-labeling could also be detected. Bars indicate 10 µm, nuclei were stained with DAPI. d Ultrathin cryosection shows morphologically distorted, elongated caveolae (arrowheads) indicating the effect of Dynasore treatment. Larger gold particles labeling ER-α could be observed in the close vicinity of caveolin-1 positive structures right beneath the plasma membrane (P). Note that the deeper cytoplasmic areas lack both small (ERα) and larger (caveolin-1) gold particles indicating the disturbed internalization of caveolin-1 positive vesicles. Bar indicates 200 nm

    Journal: European Journal of Medical Research

    Article Title: Membrane-bound estrogen receptor alpha initiated signaling is dynamin dependent in breast cancer cells

    doi: 10.1186/s40001-018-0328-7

    Figure Lengend Snippet: Morphological changes upon combined Dynasore-E2 treatment of MCF-7 cells. Semithin frozen sections of control MCF-7 cells labeled with antibodies directed against ER-α (green) and caveolin-1 (red). a – c ER-α receptor labeling occurred inside the cytoplasm and the plasma membrane in MCF-7 cells. Nuclear occurrence of the receptor could also be observed, however, to a lesser extent. The merged image of ER-α and caveolin-1 double labeling shows overlapping areas at the plasma membrane (arrowheads), while intracytoplasmic co-labeling could also be detected. Bars indicate 10 µm, nuclei were stained with DAPI. d Ultrathin cryosection shows morphologically distorted, elongated caveolae (arrowheads) indicating the effect of Dynasore treatment. Larger gold particles labeling ER-α could be observed in the close vicinity of caveolin-1 positive structures right beneath the plasma membrane (P). Note that the deeper cytoplasmic areas lack both small (ERα) and larger (caveolin-1) gold particles indicating the disturbed internalization of caveolin-1 positive vesicles. Bar indicates 200 nm

    Article Snippet: G1 (Tocris Bioscience, Bristol, UK) was dissolved in dimethyl sulfoxide (DMSO) in the final concentration of 10−8 M. Dynasore (Sigma-Aldrich) was dissolved in DMSO and used in 80 µmol treatment concentration, and was applied 30 min prior to subsequent 17β-estradiol treatment (in 10−10 M concentration).

    Techniques: Labeling, Staining

    Gene expression changes after treatment of MCF-7 cells with 17β-estradiol and estrogen–BSA. Expression changes of CCND1, ERBB2, KCNK5, KDM4B, and MYC after 17-β-estradiol (E2) ( a ) and estrogen–BSA treatment ( b ). Treatments were performed in three different concentrations (10 −10 M, 10 −9 M, 10 −8 M) and a similar E2 treatment (10 −10 M) on dynamin inhibitor (dynasore, 30 min) pretreated cells. Y -axis represents ddCT values, 0 line indicates control level. (Error bars represent standard deviation, asterisks indicate significant changes compared to control with p : Table Sheet 5

    Journal: European Journal of Medical Research

    Article Title: Membrane-bound estrogen receptor alpha initiated signaling is dynamin dependent in breast cancer cells

    doi: 10.1186/s40001-018-0328-7

    Figure Lengend Snippet: Gene expression changes after treatment of MCF-7 cells with 17β-estradiol and estrogen–BSA. Expression changes of CCND1, ERBB2, KCNK5, KDM4B, and MYC after 17-β-estradiol (E2) ( a ) and estrogen–BSA treatment ( b ). Treatments were performed in three different concentrations (10 −10 M, 10 −9 M, 10 −8 M) and a similar E2 treatment (10 −10 M) on dynamin inhibitor (dynasore, 30 min) pretreated cells. Y -axis represents ddCT values, 0 line indicates control level. (Error bars represent standard deviation, asterisks indicate significant changes compared to control with p : Table Sheet 5

    Article Snippet: G1 (Tocris Bioscience, Bristol, UK) was dissolved in dimethyl sulfoxide (DMSO) in the final concentration of 10−8 M. Dynasore (Sigma-Aldrich) was dissolved in DMSO and used in 80 µmol treatment concentration, and was applied 30 min prior to subsequent 17β-estradiol treatment (in 10−10 M concentration).

    Techniques: Expressing, Standard Deviation

    A selective dynamin inhibitor Dynasore reduces βCTF and Aβ levels. A) N2a 695 cells were treated with dynasore at 10 µM or BACE Inhibitor IV at 15µM before subjected for further analysis. Levels of βCTF and Aβ were determined and significant reductions were observed upon dynasore or BACE inhibitor treatment, as compared to control (* p

    Journal: PLoS ONE

    Article Title: Dynamin 1 Regulates Amyloid Generation through Modulation of BACE-1

    doi: 10.1371/journal.pone.0045033

    Figure Lengend Snippet: A selective dynamin inhibitor Dynasore reduces βCTF and Aβ levels. A) N2a 695 cells were treated with dynasore at 10 µM or BACE Inhibitor IV at 15µM before subjected for further analysis. Levels of βCTF and Aβ were determined and significant reductions were observed upon dynasore or BACE inhibitor treatment, as compared to control (* p

    Article Snippet: Alternatively, cells were treated with a dyn inhibitor Dynasore at 10 µM (Sigma), or a BACE inhibitor at 15 µM (Inhibitor IV, EMD) overnight before subjected for further analysis.

    Techniques:

    Blood flow measurement after injection of EPCs, EPCs + αPKCi, EPCs + dynasore groups (A) and the quantitative measurement of perfusion ratio of different groups at days 0, 7, 14, 28 respectively (B). *, P

    Journal: Journal of Thoracic Disease

    Article Title: VEGFR endocytosis regulates the angiogenesis in a mouse model of hindlimb ischemia

    doi: 10.21037/jtd.2019.05.18

    Figure Lengend Snippet: Blood flow measurement after injection of EPCs, EPCs + αPKCi, EPCs + dynasore groups (A) and the quantitative measurement of perfusion ratio of different groups at days 0, 7, 14, 28 respectively (B). *, P

    Article Snippet: Then all mice were randomly divided into four groups (n=6 each): sham-treated (PBS) group, EPCs group (4×106 cells per mouse), EPCs + αPKCi (100 µM, Calbiochem, San Diego, USA) group and EPCs + dynasore (100 µM, Sigma-Aldrich, Shanghai, China) group.

    Techniques: Flow Cytometry, Injection

    VEGFR2 endocytosis after treated EPCs with αPKCi and dynasore. (A) The expression of surface VEGFR2 and total VEGFR2 after treated EPCs with dynasore, αPKCi or vehicle (0.1% DMSO) by Western blot. (B) The quantitative data of surface VEGFR2 (*, P

    Journal: Journal of Thoracic Disease

    Article Title: VEGFR endocytosis regulates the angiogenesis in a mouse model of hindlimb ischemia

    doi: 10.21037/jtd.2019.05.18

    Figure Lengend Snippet: VEGFR2 endocytosis after treated EPCs with αPKCi and dynasore. (A) The expression of surface VEGFR2 and total VEGFR2 after treated EPCs with dynasore, αPKCi or vehicle (0.1% DMSO) by Western blot. (B) The quantitative data of surface VEGFR2 (*, P

    Article Snippet: Then all mice were randomly divided into four groups (n=6 each): sham-treated (PBS) group, EPCs group (4×106 cells per mouse), EPCs + αPKCi (100 µM, Calbiochem, San Diego, USA) group and EPCs + dynasore (100 µM, Sigma-Aldrich, Shanghai, China) group.

    Techniques: Expressing, Western Blot

    Entry of HCMV laboratory strains AD169 (BAD32) ( A ) and Towne ( B ) in HF upon treatment with pitstop 2, or dynasore as determined by expression of viral immediate early 1 (IE1) protein in infected cells. Confluent HF monolayers were pretreated with dynasore (100 μM) or pitstop 2 (25 μM) for 1 h, then infected with BAD32GFP or Towne virus at an MOI of 3.0 in the medium containing the same concentration of the drug for one hour, washed and thereafter incubated for 6 hours in the presence of the same concentration of drug before harvesting for immunoblots. Triplicate samples were used. DMSO-treated infected cells, untreated infected cells (UT) and untreated uninfected cells (UI) served as controls in this experiment. β-actin was used as a loading control. Immunoblots were digitally cropped to conserve the space.

    Journal: Scientific Reports

    Article Title: Inhibition of endocytic pathways impacts cytomegalovirus maturation

    doi: 10.1038/srep46069

    Figure Lengend Snippet: Entry of HCMV laboratory strains AD169 (BAD32) ( A ) and Towne ( B ) in HF upon treatment with pitstop 2, or dynasore as determined by expression of viral immediate early 1 (IE1) protein in infected cells. Confluent HF monolayers were pretreated with dynasore (100 μM) or pitstop 2 (25 μM) for 1 h, then infected with BAD32GFP or Towne virus at an MOI of 3.0 in the medium containing the same concentration of the drug for one hour, washed and thereafter incubated for 6 hours in the presence of the same concentration of drug before harvesting for immunoblots. Triplicate samples were used. DMSO-treated infected cells, untreated infected cells (UT) and untreated uninfected cells (UI) served as controls in this experiment. β-actin was used as a loading control. Immunoblots were digitally cropped to conserve the space.

    Article Snippet: Drug inhibition experiments Confluent HF monolayers were pretreated with dynasore (#324410, Merck Millipore, Billerica, MA) or pitstop 2 (# ab120687, Abcam, Cambridge, MA) for 1 h, then infected with Towne or BAD32GFP virus at an MOI of 3.0 in medium containing drug for one hour, washed and thereafter incubated for up to 5 days in the presence of drug.

    Techniques: Expressing, Infection, Concentration Assay, Incubation, Western Blot

    Transmission electron micrographs of HF cells that were either mock-treated ( A , B and C ) or treated with dynasore (100 μM) ( D , E and F ) or pitstop 2 (25 μM) ( G , H and I ) and then infected with HCMV (AD169) at an MOI of 3.0 and incubated in the presence of indicated drug for 4 days before processing for TEM. Representative whole cell ( A , D , G ), nuclear ( B , E , H ) or cytoplasmic ( C , F , I ) sections of the infected cell are shown in the micrographs. ( J ) Quantification of nuclear capsid types from the above treatments. A total of 400, 318 or 674 capsids were counted for mock, dynasore and pitstop 2 treatments, respectively. Scale: 2 μm ( A , C , D ), 1.0 μm ( F , G ), 0.5 μm ( B , E , H , I ). A- (white arrowheads), B- (black arrows), and C- capsids (black arrowheads).

    Journal: Scientific Reports

    Article Title: Inhibition of endocytic pathways impacts cytomegalovirus maturation

    doi: 10.1038/srep46069

    Figure Lengend Snippet: Transmission electron micrographs of HF cells that were either mock-treated ( A , B and C ) or treated with dynasore (100 μM) ( D , E and F ) or pitstop 2 (25 μM) ( G , H and I ) and then infected with HCMV (AD169) at an MOI of 3.0 and incubated in the presence of indicated drug for 4 days before processing for TEM. Representative whole cell ( A , D , G ), nuclear ( B , E , H ) or cytoplasmic ( C , F , I ) sections of the infected cell are shown in the micrographs. ( J ) Quantification of nuclear capsid types from the above treatments. A total of 400, 318 or 674 capsids were counted for mock, dynasore and pitstop 2 treatments, respectively. Scale: 2 μm ( A , C , D ), 1.0 μm ( F , G ), 0.5 μm ( B , E , H , I ). A- (white arrowheads), B- (black arrows), and C- capsids (black arrowheads).

    Article Snippet: Drug inhibition experiments Confluent HF monolayers were pretreated with dynasore (#324410, Merck Millipore, Billerica, MA) or pitstop 2 (# ab120687, Abcam, Cambridge, MA) for 1 h, then infected with Towne or BAD32GFP virus at an MOI of 3.0 in medium containing drug for one hour, washed and thereafter incubated for up to 5 days in the presence of drug.

    Techniques: Transmission Assay, Infection, Incubation, Transmission Electron Microscopy

    Effect of drug treatments on cell viability. Confluent HF monolayers were pretreated with dynasore (100 μM) or pitstop 2 (25 μM) for 1 h, then infected with Towne or BAD32GFP virus at an MOI of 3.0 in the medium containing the same concentration of the drug for one hour, washed and thereafter incubated for ( A ) 6 hours or ( B ) 5 days in the presence of the same concentration of the drug. DMSO-treated infected cells and uninfected untreated cells served as controls in this experiment. Cel l viability was determined using trypan blue exclusion assay as described in materials and methods. Triplicate samples were used.

    Journal: Scientific Reports

    Article Title: Inhibition of endocytic pathways impacts cytomegalovirus maturation

    doi: 10.1038/srep46069

    Figure Lengend Snippet: Effect of drug treatments on cell viability. Confluent HF monolayers were pretreated with dynasore (100 μM) or pitstop 2 (25 μM) for 1 h, then infected with Towne or BAD32GFP virus at an MOI of 3.0 in the medium containing the same concentration of the drug for one hour, washed and thereafter incubated for ( A ) 6 hours or ( B ) 5 days in the presence of the same concentration of the drug. DMSO-treated infected cells and uninfected untreated cells served as controls in this experiment. Cel l viability was determined using trypan blue exclusion assay as described in materials and methods. Triplicate samples were used.

    Article Snippet: Drug inhibition experiments Confluent HF monolayers were pretreated with dynasore (#324410, Merck Millipore, Billerica, MA) or pitstop 2 (# ab120687, Abcam, Cambridge, MA) for 1 h, then infected with Towne or BAD32GFP virus at an MOI of 3.0 in medium containing drug for one hour, washed and thereafter incubated for up to 5 days in the presence of drug.

    Techniques: Infection, Concentration Assay, Incubation, Trypan Blue Exclusion Assay

    Pitstop 2 or dynasore treatment does not eliminate the expression of viral early to late proteins in infected cells. Confluent HF monolayers were pretreated with dynasore (100 μM), pitstop 2 (25 μM), or mock (DMSO) for 1 h, then infected with BAD32GFP virus at an MOI of 3.0 in the medium containing the same concentration of the drug for one hour, washed and thereafter incubated for 72 hours in the presence of the same concentration of drug before harvesting for immunoblots using antibodies against early (pUL44) and late (gB) viral antigens. Duplicate samples were used for each treatment. DMSO-treated infected cells served as mock-control in this experiment and β-actin was used as a loading control. Immunoblots were digitally cropped to conserve the space.

    Journal: Scientific Reports

    Article Title: Inhibition of endocytic pathways impacts cytomegalovirus maturation

    doi: 10.1038/srep46069

    Figure Lengend Snippet: Pitstop 2 or dynasore treatment does not eliminate the expression of viral early to late proteins in infected cells. Confluent HF monolayers were pretreated with dynasore (100 μM), pitstop 2 (25 μM), or mock (DMSO) for 1 h, then infected with BAD32GFP virus at an MOI of 3.0 in the medium containing the same concentration of the drug for one hour, washed and thereafter incubated for 72 hours in the presence of the same concentration of drug before harvesting for immunoblots using antibodies against early (pUL44) and late (gB) viral antigens. Duplicate samples were used for each treatment. DMSO-treated infected cells served as mock-control in this experiment and β-actin was used as a loading control. Immunoblots were digitally cropped to conserve the space.

    Article Snippet: Drug inhibition experiments Confluent HF monolayers were pretreated with dynasore (#324410, Merck Millipore, Billerica, MA) or pitstop 2 (# ab120687, Abcam, Cambridge, MA) for 1 h, then infected with Towne or BAD32GFP virus at an MOI of 3.0 in medium containing drug for one hour, washed and thereafter incubated for up to 5 days in the presence of drug.

    Techniques: Expressing, Infection, Concentration Assay, Incubation, Western Blot

    Inhibition of endocytosis impacts the growth of HCMV laboratory strains (Towne and AD169 (BAD32GFP)) in fibroblasts. Confluent HF monolayers were pretreated with dynasore, or pitstop 2 for 1 h, and then infected with Towne or BAD32GFP virus at an MOI of 3.0 in the medium containing the same drug for one hour, washed and thereafter incubated in the presence of the drug. Samples of infected-cells in the cell culture medium were harvested at 5 days post infection and stored at −80 °C before titration. Concentration of pitstop 2 ( A ) in the range of zero to 25 μM or of dynasore ( B ) in the range of zero to 250 μM led to dose dependent inhibition of final virus yields. Triplicate samples were used.

    Journal: Scientific Reports

    Article Title: Inhibition of endocytic pathways impacts cytomegalovirus maturation

    doi: 10.1038/srep46069

    Figure Lengend Snippet: Inhibition of endocytosis impacts the growth of HCMV laboratory strains (Towne and AD169 (BAD32GFP)) in fibroblasts. Confluent HF monolayers were pretreated with dynasore, or pitstop 2 for 1 h, and then infected with Towne or BAD32GFP virus at an MOI of 3.0 in the medium containing the same drug for one hour, washed and thereafter incubated in the presence of the drug. Samples of infected-cells in the cell culture medium were harvested at 5 days post infection and stored at −80 °C before titration. Concentration of pitstop 2 ( A ) in the range of zero to 25 μM or of dynasore ( B ) in the range of zero to 250 μM led to dose dependent inhibition of final virus yields. Triplicate samples were used.

    Article Snippet: Drug inhibition experiments Confluent HF monolayers were pretreated with dynasore (#324410, Merck Millipore, Billerica, MA) or pitstop 2 (# ab120687, Abcam, Cambridge, MA) for 1 h, then infected with Towne or BAD32GFP virus at an MOI of 3.0 in medium containing drug for one hour, washed and thereafter incubated for up to 5 days in the presence of drug.

    Techniques: Inhibition, Infection, Incubation, Cell Culture, Titration, Concentration Assay

    Co-localization of clathrin and pp150 during HCMV infection when cells were dynasore treated at infection or at consecutive days post infection. HF were mock-infected or infected with BAD32GFP virus at an MOI of 3.0 and fixed for IFA at 5 days post infection. Dynasore was added at day 1 (at the time of infection) or at days 2, 3, 4 of infection. Shown are the groups of four panels (mock or infected), obtained from the same field that includes single-color images of the cellular antigen (red), viral antigen pp150 (green), DNA (in nuclei) detected by Hoechst 33258 (blue), or a composite (overlay, right). Clathrin heavy chain antibody was used for clathrin detection and the GFP fluorescence from BAD32GFP virus marked pp150. vAC are marked with an arrow in the overlay image.

    Journal: Scientific Reports

    Article Title: Inhibition of endocytic pathways impacts cytomegalovirus maturation

    doi: 10.1038/srep46069

    Figure Lengend Snippet: Co-localization of clathrin and pp150 during HCMV infection when cells were dynasore treated at infection or at consecutive days post infection. HF were mock-infected or infected with BAD32GFP virus at an MOI of 3.0 and fixed for IFA at 5 days post infection. Dynasore was added at day 1 (at the time of infection) or at days 2, 3, 4 of infection. Shown are the groups of four panels (mock or infected), obtained from the same field that includes single-color images of the cellular antigen (red), viral antigen pp150 (green), DNA (in nuclei) detected by Hoechst 33258 (blue), or a composite (overlay, right). Clathrin heavy chain antibody was used for clathrin detection and the GFP fluorescence from BAD32GFP virus marked pp150. vAC are marked with an arrow in the overlay image.

    Article Snippet: Drug inhibition experiments Confluent HF monolayers were pretreated with dynasore (#324410, Merck Millipore, Billerica, MA) or pitstop 2 (# ab120687, Abcam, Cambridge, MA) for 1 h, then infected with Towne or BAD32GFP virus at an MOI of 3.0 in medium containing drug for one hour, washed and thereafter incubated for up to 5 days in the presence of drug.

    Techniques: Infection, Immunofluorescence, Fluorescence

    Inhibition of DNM decreases nitrite production. A : nitrite production from COS7 cells overexpressing NOS1α + DNM2 was inhibited by the dynamin inhibitor dynasore in the presence and absence of ionomycin. Statistical analysis by 2-way ANOVA. *

    Journal: American Journal of Physiology - Renal Physiology

    Article Title: Dynamin activates NO production in rat renal inner medullary collecting ducts via protein-protein interaction with NOS1

    doi: 10.1152/ajprenal.00534.2010

    Figure Lengend Snippet: Inhibition of DNM decreases nitrite production. A : nitrite production from COS7 cells overexpressing NOS1α + DNM2 was inhibited by the dynamin inhibitor dynasore in the presence and absence of ionomycin. Statistical analysis by 2-way ANOVA. *

    Article Snippet: COS7 cells transfected with NOS1α and DNM2-GFP were grown to confluency, serum-starved 4 h, and treated with the DNM-specific inhibitor dynasore ( ) (Sigma).

    Techniques: Inhibition

    Osteoblast differentiation is arrested by inhibition of dynamin-dependent endocytosis. ( A,B ) Confluent C2C12 cells were serum-starved for 2 h prior to stimulation with the indicated concentrations of BMP-2. After 72 h of stimulation, cells were lysed and ALP activity was measured at 405 nm by conversion of para-nitrophenylphosphate. A schematic representation of the treatment is depicted above the respective histogram. The histograms show mean ± s.d. of triplicate measurements representative of three independent experiments. ( A ) During starvation and initial 4 h of stimulation, 40 µM dynasore or 0.05% DSMO were added. (P-values in relation to control treated samples). ( B ) After 4 h of stimulation, BMP-2-containing medium was replaced by medium without BMP-2. (** P-values in relation to unstimulated samples; 30 nM: P = 0.008, 60 nM: P = 0.0017) ( C–G ) Confluent C2C12 cells were treated as described in (A) with 30 nM BMP-2. Total RNA was isolated, cDNA was synthesized and mRNA expression was analyzed by qPCR using specific mouse primers ( Table S1 ). Histograms show mean normalized expression (MNE) with standard error of duplicate measurements relative to the housekeeping gene GAPDH . The results are representative of three independent experiments.

    Journal: PLoS ONE

    Article Title: Spatial Segregation of BMP/Smad Signaling Affects Osteoblast Differentiation in C2C12 Cells

    doi: 10.1371/journal.pone.0025163

    Figure Lengend Snippet: Osteoblast differentiation is arrested by inhibition of dynamin-dependent endocytosis. ( A,B ) Confluent C2C12 cells were serum-starved for 2 h prior to stimulation with the indicated concentrations of BMP-2. After 72 h of stimulation, cells were lysed and ALP activity was measured at 405 nm by conversion of para-nitrophenylphosphate. A schematic representation of the treatment is depicted above the respective histogram. The histograms show mean ± s.d. of triplicate measurements representative of three independent experiments. ( A ) During starvation and initial 4 h of stimulation, 40 µM dynasore or 0.05% DSMO were added. (P-values in relation to control treated samples). ( B ) After 4 h of stimulation, BMP-2-containing medium was replaced by medium without BMP-2. (** P-values in relation to unstimulated samples; 30 nM: P = 0.008, 60 nM: P = 0.0017) ( C–G ) Confluent C2C12 cells were treated as described in (A) with 30 nM BMP-2. Total RNA was isolated, cDNA was synthesized and mRNA expression was analyzed by qPCR using specific mouse primers ( Table S1 ). Histograms show mean normalized expression (MNE) with standard error of duplicate measurements relative to the housekeeping gene GAPDH . The results are representative of three independent experiments.

    Article Snippet: The endocytosis inhibitor dynasore (Sigma) was aliquoted under argon as a stock solution of 80 mM in DMSO and kept light protected at −80°C .

    Techniques: Inhibition, ALP Assay, Activity Assay, Isolation, Synthesized, Expressing, Real-time Polymerase Chain Reaction

    Smad1/5/8 nuclear translocation is delayed and transcriptional activity is reduced by inhibition of dynamin-dependent endocytosis. ( A ) Serum-starved C2C12 cells were treated for 30 min with 40 µM dynasore or 0.05% DMSO prior to stimulation with 10 nM BMP-2 for 30 min in medium containing dynasore or DMSO. After fixation, endogenous Smad1 was stained using a specific antibody, nuclei were stained by Hoechst dye, and cells were analyzed by fluorescence microscopy. The panels shown are representative of two independent experiments. Bar, 10 µm. ( B ) Quantification of the experiment shown in (A). Relative fluorescence intensity of nuclear to cytoplasmic Smad1 staining is depicted in the histogram. The results are mean ± s.d. of at least 100 cells. AU, arbitrary units. ( C ) Serum-starved C2C12 cells were treated for 2 h with 40 µM dynasore or 0.05% DSMO prior to stimulation with 10 nM BMP-2 for the indicated time periods in medium containing dynasore or DMSO. Samples were subjected to cytoplasmic-nuclear fractionation and processed for immunoblotting with anti-phospho-specific Smad1/5/8 antibody (anti-pSmad1/5/8). To control fractionation, samples were analyzed using anti-GAPDH or anti-Histon H3 antibodies. The Western Blot is representative of two independent experiments. c, cytosol; n, nucleus. ( D,E ) C2C12 cells were co-transfected with BRE-Luc and RL-TK. Cells were serum-starved and treated with 40 µM dynasore or 0.05% DMSO for 1 h prior to stimulation with 3 nM BMP-2 for 6 h (D) or 24 h (E) in medium containing dynasore or DMSO. Relative luciferase activity (RLA) of BRE-driven luciferase compared to constitutive expression of RL-TK is shown. Results are mean ± s.d. of triplicate measurements, representative of three independent experiments.

    Journal: PLoS ONE

    Article Title: Spatial Segregation of BMP/Smad Signaling Affects Osteoblast Differentiation in C2C12 Cells

    doi: 10.1371/journal.pone.0025163

    Figure Lengend Snippet: Smad1/5/8 nuclear translocation is delayed and transcriptional activity is reduced by inhibition of dynamin-dependent endocytosis. ( A ) Serum-starved C2C12 cells were treated for 30 min with 40 µM dynasore or 0.05% DMSO prior to stimulation with 10 nM BMP-2 for 30 min in medium containing dynasore or DMSO. After fixation, endogenous Smad1 was stained using a specific antibody, nuclei were stained by Hoechst dye, and cells were analyzed by fluorescence microscopy. The panels shown are representative of two independent experiments. Bar, 10 µm. ( B ) Quantification of the experiment shown in (A). Relative fluorescence intensity of nuclear to cytoplasmic Smad1 staining is depicted in the histogram. The results are mean ± s.d. of at least 100 cells. AU, arbitrary units. ( C ) Serum-starved C2C12 cells were treated for 2 h with 40 µM dynasore or 0.05% DSMO prior to stimulation with 10 nM BMP-2 for the indicated time periods in medium containing dynasore or DMSO. Samples were subjected to cytoplasmic-nuclear fractionation and processed for immunoblotting with anti-phospho-specific Smad1/5/8 antibody (anti-pSmad1/5/8). To control fractionation, samples were analyzed using anti-GAPDH or anti-Histon H3 antibodies. The Western Blot is representative of two independent experiments. c, cytosol; n, nucleus. ( D,E ) C2C12 cells were co-transfected with BRE-Luc and RL-TK. Cells were serum-starved and treated with 40 µM dynasore or 0.05% DMSO for 1 h prior to stimulation with 3 nM BMP-2 for 6 h (D) or 24 h (E) in medium containing dynasore or DMSO. Relative luciferase activity (RLA) of BRE-driven luciferase compared to constitutive expression of RL-TK is shown. Results are mean ± s.d. of triplicate measurements, representative of three independent experiments.

    Article Snippet: The endocytosis inhibitor dynasore (Sigma) was aliquoted under argon as a stock solution of 80 mM in DMSO and kept light protected at −80°C .

    Techniques: Translocation Assay, Activity Assay, Inhibition, Staining, Fluorescence, Microscopy, Fractionation, Western Blot, Transfection, Luciferase, Expressing

    Validation of two classes of genes, which are dependent or independent of endocytosis. ( A,B ) Serum-starved C2C12 cells were treated for 2 h with 40 µM dynasore or 0.05% DMSO prior to stimulation with 30 nM BMP-2 for 6 h in medium containing dynasore or DMSO. Total RNA was isolated, cDNA was synthesized and mRNA expression was analyzed by qPCR using specific mouse primers ( Table S1 ). Histograms show mean normalized expression (MNE) with standard error of duplicate measurements relative to the housekeeping gene HPRT . The analysis shown is representative of three independent experiments. ( C ) C2C12 cells were co-transfected with Id1-Luc or Id2-Luc and RL-TK. Cells were serum-starved and treated with 40 µM dynasore or 0.05% DMSO for 1 h prior to stimulation with 3 nM BMP-2 for 24 h in medium containing dynasore or DMSO. Relative luciferase activity (RLA) of Id1- or Id2-driven luciferase compared to constitutive expression of RL-TK is shown. Results are shown as mean ± s.d. of triplicate measurements, representative of three independent experiments.

    Journal: PLoS ONE

    Article Title: Spatial Segregation of BMP/Smad Signaling Affects Osteoblast Differentiation in C2C12 Cells

    doi: 10.1371/journal.pone.0025163

    Figure Lengend Snippet: Validation of two classes of genes, which are dependent or independent of endocytosis. ( A,B ) Serum-starved C2C12 cells were treated for 2 h with 40 µM dynasore or 0.05% DMSO prior to stimulation with 30 nM BMP-2 for 6 h in medium containing dynasore or DMSO. Total RNA was isolated, cDNA was synthesized and mRNA expression was analyzed by qPCR using specific mouse primers ( Table S1 ). Histograms show mean normalized expression (MNE) with standard error of duplicate measurements relative to the housekeeping gene HPRT . The analysis shown is representative of three independent experiments. ( C ) C2C12 cells were co-transfected with Id1-Luc or Id2-Luc and RL-TK. Cells were serum-starved and treated with 40 µM dynasore or 0.05% DMSO for 1 h prior to stimulation with 3 nM BMP-2 for 24 h in medium containing dynasore or DMSO. Relative luciferase activity (RLA) of Id1- or Id2-driven luciferase compared to constitutive expression of RL-TK is shown. Results are shown as mean ± s.d. of triplicate measurements, representative of three independent experiments.

    Article Snippet: The endocytosis inhibitor dynasore (Sigma) was aliquoted under argon as a stock solution of 80 mM in DMSO and kept light protected at −80°C .

    Techniques: Isolation, Synthesized, Expressing, Real-time Polymerase Chain Reaction, Transfection, Luciferase, Activity Assay

    Phosphorylation of Smad1/5/8 is delayed by inhibition of dynamin-dependent endocytosis. ( A ) Serum starved C2C12 cells were treated for 2 h with 40 µM dynasore or 0.05% DMSO and incubated for 15 min at 37°C with Alexa594-transferrin in the presence of dynasore and DMSO. Cells were fixed and transferrin uptake was monitored by fluorescence microscopy. Lower panels show Hoechst staining of the respective cells. Bar, 10 µm. ( B ) Quantification of transferrin uptake shown in (A). The histogram depicts the amount of internalized transferrin, derived from the total fluorescence signal of Alexa594-transferrin within the cell boundaries. The results are mean ± s.d. of at least 400 cells. AU, arbitrary units. ( C ) Serum-starved C2C12 cells were treated for 2 h with 40 µM dynasore or 0.05% DSMO prior to stimulation with 10 nM BMP-2 for the indicated time periods in medium containing dynasore or DMSO. Samples were processed for immunoblotting with anti-phospho-specific Smad1/5/8 antibody (anti-pSmad1/5/8) or GAPDH antibody (anti-GAPDH) as loading control. The Western Blot shown is representative of three independent experiments.

    Journal: PLoS ONE

    Article Title: Spatial Segregation of BMP/Smad Signaling Affects Osteoblast Differentiation in C2C12 Cells

    doi: 10.1371/journal.pone.0025163

    Figure Lengend Snippet: Phosphorylation of Smad1/5/8 is delayed by inhibition of dynamin-dependent endocytosis. ( A ) Serum starved C2C12 cells were treated for 2 h with 40 µM dynasore or 0.05% DMSO and incubated for 15 min at 37°C with Alexa594-transferrin in the presence of dynasore and DMSO. Cells were fixed and transferrin uptake was monitored by fluorescence microscopy. Lower panels show Hoechst staining of the respective cells. Bar, 10 µm. ( B ) Quantification of transferrin uptake shown in (A). The histogram depicts the amount of internalized transferrin, derived from the total fluorescence signal of Alexa594-transferrin within the cell boundaries. The results are mean ± s.d. of at least 400 cells. AU, arbitrary units. ( C ) Serum-starved C2C12 cells were treated for 2 h with 40 µM dynasore or 0.05% DSMO prior to stimulation with 10 nM BMP-2 for the indicated time periods in medium containing dynasore or DMSO. Samples were processed for immunoblotting with anti-phospho-specific Smad1/5/8 antibody (anti-pSmad1/5/8) or GAPDH antibody (anti-GAPDH) as loading control. The Western Blot shown is representative of three independent experiments.

    Article Snippet: The endocytosis inhibitor dynasore (Sigma) was aliquoted under argon as a stock solution of 80 mM in DMSO and kept light protected at −80°C .

    Techniques: Inhibition, Incubation, Fluorescence, Microscopy, Staining, Derivative Assay, Western Blot

    Gene expression is differentially affected by inhibition of dynamin-dependent endocytosis. Serum-starved C2C12 cells were treated for 2 h with 40 µM dynasore or 0.05% DSMO prior to stimulation with 30 nM BMP-2 for 6 h in medium containing dynasore or DMSO. RNA was isolated and subjected to whole genome profiling using the Illumina BeadChip system. Expression profiles were analyzed relative to DMSO control treatment. ( A ) Venn diagram based on significantly detected genes (1.4 fold regulation, detection P-value

    Journal: PLoS ONE

    Article Title: Spatial Segregation of BMP/Smad Signaling Affects Osteoblast Differentiation in C2C12 Cells

    doi: 10.1371/journal.pone.0025163

    Figure Lengend Snippet: Gene expression is differentially affected by inhibition of dynamin-dependent endocytosis. Serum-starved C2C12 cells were treated for 2 h with 40 µM dynasore or 0.05% DSMO prior to stimulation with 30 nM BMP-2 for 6 h in medium containing dynasore or DMSO. RNA was isolated and subjected to whole genome profiling using the Illumina BeadChip system. Expression profiles were analyzed relative to DMSO control treatment. ( A ) Venn diagram based on significantly detected genes (1.4 fold regulation, detection P-value

    Article Snippet: The endocytosis inhibitor dynasore (Sigma) was aliquoted under argon as a stock solution of 80 mM in DMSO and kept light protected at −80°C .

    Techniques: Expressing, Inhibition, Isolation

    Macrophage endocytosis of HMGB1 is mediated by RAGE-dependent pathway. ( a ) Confocal microscopy of alveolar macrophages (AMs) that were isolated from C57BL/6 (wild type, WT) mice and incubated with recombinant protein HMGB1-EGFP (100 nmol/l) or EGFP for 0–30 min. Original magnification × 600; results are representative of three independent experiments. ( b ) Confocal microscopy of AMs that were isolated from WT mice and incubated with 0–100 nmol/l HMGB1-EGFP for 30 min. Original magnification × 600; results are representative of three independent experiments. ( c ) Confocal microscopy of AMs that were isolated from WT, TLR2 −/− , TLR4 −/− , or RAGE −/− mice and incubated with HMGB1-EGFP (100 nmol/l) for 30 min. Original magnification × 600; results are representative of three independent experiments. ( d ) Changes in average number of intracellular EGFP-tagged protein particles in AMs, which were calculated using confocal microscopy program. The AMs were isolated from WT, TLR2 −/− , TLR4 −/− , or RAGE −/− mice and incubated with HMGB1-EGFP (100 nmol/l) for 30 min. In some experiments, WT AMs were incubated with HMGB1-EGFP and dynamin inhibitor dynasore (30 μ g/ml) for 30 min. The graph shows the mean and S.D., n =9. * P

    Journal: Cell Death and Differentiation

    Article Title: Macrophage endocytosis of high-mobility group box 1 triggers pyroptosis

    doi: 10.1038/cdd.2014.40

    Figure Lengend Snippet: Macrophage endocytosis of HMGB1 is mediated by RAGE-dependent pathway. ( a ) Confocal microscopy of alveolar macrophages (AMs) that were isolated from C57BL/6 (wild type, WT) mice and incubated with recombinant protein HMGB1-EGFP (100 nmol/l) or EGFP for 0–30 min. Original magnification × 600; results are representative of three independent experiments. ( b ) Confocal microscopy of AMs that were isolated from WT mice and incubated with 0–100 nmol/l HMGB1-EGFP for 30 min. Original magnification × 600; results are representative of three independent experiments. ( c ) Confocal microscopy of AMs that were isolated from WT, TLR2 −/− , TLR4 −/− , or RAGE −/− mice and incubated with HMGB1-EGFP (100 nmol/l) for 30 min. Original magnification × 600; results are representative of three independent experiments. ( d ) Changes in average number of intracellular EGFP-tagged protein particles in AMs, which were calculated using confocal microscopy program. The AMs were isolated from WT, TLR2 −/− , TLR4 −/− , or RAGE −/− mice and incubated with HMGB1-EGFP (100 nmol/l) for 30 min. In some experiments, WT AMs were incubated with HMGB1-EGFP and dynamin inhibitor dynasore (30 μ g/ml) for 30 min. The graph shows the mean and S.D., n =9. * P

    Article Snippet: In contrast, we observed that treatment of the cells with dynamin inhibitor dynasore (30 μ g/ml in 0.3% DMSO, Sigma-Aldrich, St. Louis, MO, USA) effectively blocked HMGB1 internalization as shown in .

    Techniques: Confocal Microscopy, Affinity Magnetic Separation, Isolation, Mouse Assay, Incubation, Recombinant

    FLNa-dependent HCN1 clustering and I h inhibition are dynamin-dependent and reversible. A, confocal images of HEK293 cells co-expressing HCN1 GFP and FLNa DsRed . HCN1 clustering was reduced following incubation with the selective dynamin-inhibitor dynasore. The images are a stacked representation of optical slices (z) with

    Journal: The Journal of Biological Chemistry

    Article Title: Filamin A Promotes Dynamin-dependent Internalization of Hyperpolarization-activated Cyclic Nucleotide-gated Type 1 (HCN1) Channels and Restricts Ih in Hippocampal Neurons *

    doi: 10.1074/jbc.M113.522060

    Figure Lengend Snippet: FLNa-dependent HCN1 clustering and I h inhibition are dynamin-dependent and reversible. A, confocal images of HEK293 cells co-expressing HCN1 GFP and FLNa DsRed . HCN1 clustering was reduced following incubation with the selective dynamin-inhibitor dynasore. The images are a stacked representation of optical slices (z) with

    Article Snippet: Inhibition of dynamin in HEK293 cells was carried out by a 4-h incubation with 80 μ m of the selective dynamin inhibitor dynasore (Sigma) ( ) at 37 °C.

    Techniques: Inhibition, Expressing, Incubation

    Cellular response to CDCs and protection by Dynasore. A ) PLO pore formation is dependent on the presence of membrane cholesterol. Cholesterol binding initiates significant secondary and tertiary structural changes in CDCs, which lead to the assembly of a large membrane-embedded β -barrel pore complex. Sublytic concentrations of PLO activated the MAPK and autophagy pathways, but these were unable to protect stromal cells against higher concentrations of toxins. B ) Dynamin GTPase inhibitor Dynasore as well as M β CD protected against stromal cytolysis caused by decreasing cellular cholesterol concentration.

    Journal: The FASEB Journal

    Article Title: Protective role of the dynamin inhibitor Dynasore against the cholesterol-dependent cytolysin of Trueperella pyogenes

    doi: 10.1096/fj.14-265207

    Figure Lengend Snippet: Cellular response to CDCs and protection by Dynasore. A ) PLO pore formation is dependent on the presence of membrane cholesterol. Cholesterol binding initiates significant secondary and tertiary structural changes in CDCs, which lead to the assembly of a large membrane-embedded β -barrel pore complex. Sublytic concentrations of PLO activated the MAPK and autophagy pathways, but these were unable to protect stromal cells against higher concentrations of toxins. B ) Dynamin GTPase inhibitor Dynasore as well as M β CD protected against stromal cytolysis caused by decreasing cellular cholesterol concentration.

    Article Snippet: The ERK1/2 inhibitor peptide (#328000), p38 inhibitor SB203580 (#559398), JNK inhibitor II (#420128), MEK1/2 inhibitor UO126 (#662005), MEK1 inhibitor PD098059 (#513001), and dynamin inhibitor 1 Dynasore (#324410) were all purchased from Calbiochem (Nottingham, United Kingdom).

    Techniques: Binding Assay, Concentration Assay

    Tat induces TLR4 down modulation by specific dynamin endocytosis. ( A ) U937, HEK-TLR4-MD2-CD14 or human monocytes were stimulated or not with 10 or 100 nM of GST +/-Tat for the indicated times. Surface expression of TLR4 in unstimulated (black line) or stimulated (pink, blue and red lines) cells was analyzed by flow cytometry using anti-TLR4 or isotype control IgG (tinted), stained with secondary FITC or PE antibodies. Data represent one of three independent experiments. ( B ) U937 were pre-incubated for 1 h with 1 μg/mL of blocking anti-TLR4 or ( C ) HEK TLR4-CD14-MD2 were pretreated with 80 μM of dynasore for 30 minutes before stimulation with GST+/-Tat (100 nM) for the time periods indicated. Cell surface expression of TLR4 was then analyzed as described above. ( D – G ) The integrity of the raft structure is necessary for Tat-induced IL-6 and IL-8: D-E ) Purified human monocytes (10 6 ) were pretreated with increasing amounts of raft disruption drug M-β-CD (10, 60 min) or F-G ) with dynasore for 30 minutes before stimulation with GST-Tat 1–101 (100 nM). After 24 h, IL-6 and IL-8 production in the culture supernatants were quantified by ELISA. The data represent means (pg/ml) and SD (n > 3). As controls, cells were stimulated with PBS, DMSO or the highest drug concentration and cytokine production and cytotoxicity (trypan blue) were analyzed. Asterisks represent P values: *, P ˂ 0.05; **, P ˂ 0.01; ***, P ˂ 0.001, ns non significant.

    Journal: PLoS ONE

    Article Title: HIV-1 Tat Protein Induces Production of Proinflammatory Cytokines by Human Dendritic Cells and Monocytes/Macrophages through Engagement of TLR4-MD2-CD14 Complex and Activation of NF-κB Pathway

    doi: 10.1371/journal.pone.0129425

    Figure Lengend Snippet: Tat induces TLR4 down modulation by specific dynamin endocytosis. ( A ) U937, HEK-TLR4-MD2-CD14 or human monocytes were stimulated or not with 10 or 100 nM of GST +/-Tat for the indicated times. Surface expression of TLR4 in unstimulated (black line) or stimulated (pink, blue and red lines) cells was analyzed by flow cytometry using anti-TLR4 or isotype control IgG (tinted), stained with secondary FITC or PE antibodies. Data represent one of three independent experiments. ( B ) U937 were pre-incubated for 1 h with 1 μg/mL of blocking anti-TLR4 or ( C ) HEK TLR4-CD14-MD2 were pretreated with 80 μM of dynasore for 30 minutes before stimulation with GST+/-Tat (100 nM) for the time periods indicated. Cell surface expression of TLR4 was then analyzed as described above. ( D – G ) The integrity of the raft structure is necessary for Tat-induced IL-6 and IL-8: D-E ) Purified human monocytes (10 6 ) were pretreated with increasing amounts of raft disruption drug M-β-CD (10, 60 min) or F-G ) with dynasore for 30 minutes before stimulation with GST-Tat 1–101 (100 nM). After 24 h, IL-6 and IL-8 production in the culture supernatants were quantified by ELISA. The data represent means (pg/ml) and SD (n > 3). As controls, cells were stimulated with PBS, DMSO or the highest drug concentration and cytokine production and cytotoxicity (trypan blue) were analyzed. Asterisks represent P values: *, P ˂ 0.05; **, P ˂ 0.01; ***, P ˂ 0.001, ns non significant.

    Article Snippet: The raft disrupting drug methyl–β-cyclodextrin (M-βCD) and a GTPase dynamin inhibitor dynasore were purchased from Sigma-Aldrich.

    Techniques: Expressing, Flow Cytometry, Cytometry, Staining, Incubation, Blocking Assay, Purification, Enzyme-linked Immunosorbent Assay, Concentration Assay

    TuMV proteins VPg and CI localize to post-Golgi compartments. (A, B) Colocalization assay of TuMV fusion proteins with the early endosome marker mCherry-AtSYP61 (A) and the late endosome marker mCherry-AtARA6 (B) in Arabidopsis protoplasts. Images were taken at 20 hpt. (C, D) Effect of dynasore treatment on the subcellular localization of YFP-TuMV VPg (C) and YFP-TuMV CI (D). Protoplasts expressing YFP-TuMV VPg or YFP-TuMV CI at 16 hpt were treated with 100 µM dynasore or DMSO for 3 h and then 40 µM FM4-64 for 1 h. Scale bar = 10 μm.

    Journal: Journal of Virology

    Article Title: Dynamin-Like Proteins of Endocytosis in Plants Are Coopted by Potyviruses To Enhance Virus Infection

    doi: 10.1128/JVI.01320-18

    Figure Lengend Snippet: TuMV proteins VPg and CI localize to post-Golgi compartments. (A, B) Colocalization assay of TuMV fusion proteins with the early endosome marker mCherry-AtSYP61 (A) and the late endosome marker mCherry-AtARA6 (B) in Arabidopsis protoplasts. Images were taken at 20 hpt. (C, D) Effect of dynasore treatment on the subcellular localization of YFP-TuMV VPg (C) and YFP-TuMV CI (D). Protoplasts expressing YFP-TuMV VPg or YFP-TuMV CI at 16 hpt were treated with 100 µM dynasore or DMSO for 3 h and then 40 µM FM4-64 for 1 h. Scale bar = 10 μm.

    Article Snippet: The endocytosis chemical inhibitors dynasore (Sigma) and Tyr A23 (Sigma) were used to inhibit the endocytic pathway, as previously reported ( , ).

    Techniques: Marker, Expressing

    Optogenetic control and visualization of Ca 2+ signals with CaMello-XRs. a CaMello and CaMello-5HT 2A design. Both chimeric constructs consist of mouse melanopsin (mOpn4L), mCherry inserted into C3 and GCaMP6m added to the CT. CaMello-5HT 2A additionally has the 5-HT 2A receptor CT appended (C: intracellular loop; CT: C-terminus; H: transmembrane helix; E: extracellular loop). b Time course of light-induced Ca 2+ responses in HEK tsA201 cells. Transfected cells were visualized (mCherry, 561 nm) and Ca 2+ signals were measured (GCaMP6m, 476 + 495 nm) (images). Normalized Ca 2+ responses during 60 s of illumination (graphs), for CaMello-5HT 2A with/without addition of dynamin inhibitor Dynasore (50 µM) (mean ± s.e.m.; n = 5 dishes). Scale bar, 10 µm. c Normalized activation-dependent receptor internalization monitored via differences in membrane-localized mCherry fluorescence reduction between stimulated (476 nm + 561 nm) and unstimulated (561 nm) trials, for CaMello-5HT2A with/without addition of Dynasore (50 µM) (mean; n = 5 dishes). d Colocalization of CaMello-5HT 2A with Rab5a (-mCitrine, early endosome), Rab7a (early to late endosome) or GALT (beta-1,4-galactosyltransferase 1, trans-Golgi network) 5 min post stimulation with 476 nm light (5 min). Scale bar, 10 µm. e Averages of the calculated Pearson’s correlation coefficient for the colocalization as shown in d (box plot; one-way analysis of variance (ANOVA) and Holm-Sidak multiple comparison method; n = 6 individual cells for each colocalization pairing; *** p

    Journal: Communications Biology

    Article Title: CaMello-XR enables visualization and optogenetic control of Gq/11 signals and receptor trafficking in GPCR-specific domains

    doi: 10.1038/s42003-019-0292-y

    Figure Lengend Snippet: Optogenetic control and visualization of Ca 2+ signals with CaMello-XRs. a CaMello and CaMello-5HT 2A design. Both chimeric constructs consist of mouse melanopsin (mOpn4L), mCherry inserted into C3 and GCaMP6m added to the CT. CaMello-5HT 2A additionally has the 5-HT 2A receptor CT appended (C: intracellular loop; CT: C-terminus; H: transmembrane helix; E: extracellular loop). b Time course of light-induced Ca 2+ responses in HEK tsA201 cells. Transfected cells were visualized (mCherry, 561 nm) and Ca 2+ signals were measured (GCaMP6m, 476 + 495 nm) (images). Normalized Ca 2+ responses during 60 s of illumination (graphs), for CaMello-5HT 2A with/without addition of dynamin inhibitor Dynasore (50 µM) (mean ± s.e.m.; n = 5 dishes). Scale bar, 10 µm. c Normalized activation-dependent receptor internalization monitored via differences in membrane-localized mCherry fluorescence reduction between stimulated (476 nm + 561 nm) and unstimulated (561 nm) trials, for CaMello-5HT2A with/without addition of Dynasore (50 µM) (mean; n = 5 dishes). d Colocalization of CaMello-5HT 2A with Rab5a (-mCitrine, early endosome), Rab7a (early to late endosome) or GALT (beta-1,4-galactosyltransferase 1, trans-Golgi network) 5 min post stimulation with 476 nm light (5 min). Scale bar, 10 µm. e Averages of the calculated Pearson’s correlation coefficient for the colocalization as shown in d (box plot; one-way analysis of variance (ANOVA) and Holm-Sidak multiple comparison method; n = 6 individual cells for each colocalization pairing; *** p

    Article Snippet: For CaMello-5HT2A control experiments Dynasore (50 µM, Sigma-Aldrich) was applied 30 min before measuring.

    Techniques: Construct, Transfection, Activation Assay, Fluorescence

    Different effects of dynasore on the replication of HIV-1(VSV-G) and Wt in a human T cell, Rev-CEM. (A) Rev-CEM, a Rev-dependent GFP indicator cell, was pretreated for 30 minutes with 80 µM, 8 µM, or 0.8 µM dynasore, respectively, or treated with 0.1% DMSO as a control. Cells were subsequently infected with HIV-1(VSV-G) in the presence of dynasore for 2 hours. Following infection, cells were washed three times with medium, and then cultured in the absence of dynasore. Viral replication was monitored by flow cytometry analysis of HIV-dependent GFP expression at 48 hours (20,000 cells analyzed per sample). Propidum iodide (PI) was added into the cell suspension prior to flow cytometry. Viable cells were gated (R1) based on low PI staining and cell size (FSC). GFP expression within the viable cell population (R1) was measured. Both the GFP percentage (%) and mean intensity (M) were shown. (B) is an identical experiment using HIV-1 NL4-3 (Wt). For the 80 µM dynasore treatment, another three-independent infections with each virus were performed. The averages from the three-independent experiments are: 15.05%±0.21 (VSV-G), 10.11%±0.06 (VSV-G plus 80 µM dynasore), p = 0.0003; 2.94%±0.39 (Wt), 2.84%±0.30 (Wt plus 80 µM dynasore), p = 0.38.

    Journal: PLoS Pathogens

    Article Title: The HIV Envelope but Not VSV Glycoprotein Is Capable of Mediating HIV Latent Infection of Resting CD4 T Cells

    doi: 10.1371/journal.ppat.1000633

    Figure Lengend Snippet: Different effects of dynasore on the replication of HIV-1(VSV-G) and Wt in a human T cell, Rev-CEM. (A) Rev-CEM, a Rev-dependent GFP indicator cell, was pretreated for 30 minutes with 80 µM, 8 µM, or 0.8 µM dynasore, respectively, or treated with 0.1% DMSO as a control. Cells were subsequently infected with HIV-1(VSV-G) in the presence of dynasore for 2 hours. Following infection, cells were washed three times with medium, and then cultured in the absence of dynasore. Viral replication was monitored by flow cytometry analysis of HIV-dependent GFP expression at 48 hours (20,000 cells analyzed per sample). Propidum iodide (PI) was added into the cell suspension prior to flow cytometry. Viable cells were gated (R1) based on low PI staining and cell size (FSC). GFP expression within the viable cell population (R1) was measured. Both the GFP percentage (%) and mean intensity (M) were shown. (B) is an identical experiment using HIV-1 NL4-3 (Wt). For the 80 µM dynasore treatment, another three-independent infections with each virus were performed. The averages from the three-independent experiments are: 15.05%±0.21 (VSV-G), 10.11%±0.06 (VSV-G plus 80 µM dynasore), p = 0.0003; 2.94%±0.39 (Wt), 2.84%±0.30 (Wt plus 80 µM dynasore), p = 0.38.

    Article Snippet: Flow cytometry Dynasore monohydrate (Sigma) was dissolved in DMSO.

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

    Luciferase transfection of LPD (LP a D, LP b D, LP c D, and LP d D) and HLPD (HLP a D, HLP b D, HLP c D, and HLP d D) in SMMC-7721 cells incubated for 24 h. Notes: In the absence (normal group) and presence (dynasore group) of ( A ) anti-CD44 antibody (50 μg/mL) pretreated for 1 h, ( B ) dynamin inhibitor I with 0.2% DMSO in the solution (DI; dynasore, 80 μM) pretreated for 1 h, ( C ) chlorpromazine (CPZ; 20 μM) pretreated for 1 h, and ( D ) 5-( N -ethyl- N -isopropyl) amiloride (EIPA; 100 μM) with 0.1% DMSO in the solution pretreated for 30 min. Values are the means of four replicates ± SD. “*” Represents an alpha value of P

    Journal: International Journal of Nanomedicine

    Article Title: Effect of inserted spacer in hepatic cell-penetrating multifunctional peptide component on the DNA intracellular delivery of quaternary complexes based on modular design

    doi: 10.2147/IJN.S115381

    Figure Lengend Snippet: Luciferase transfection of LPD (LP a D, LP b D, LP c D, and LP d D) and HLPD (HLP a D, HLP b D, HLP c D, and HLP d D) in SMMC-7721 cells incubated for 24 h. Notes: In the absence (normal group) and presence (dynasore group) of ( A ) anti-CD44 antibody (50 μg/mL) pretreated for 1 h, ( B ) dynamin inhibitor I with 0.2% DMSO in the solution (DI; dynasore, 80 μM) pretreated for 1 h, ( C ) chlorpromazine (CPZ; 20 μM) pretreated for 1 h, and ( D ) 5-( N -ethyl- N -isopropyl) amiloride (EIPA; 100 μM) with 0.1% DMSO in the solution pretreated for 30 min. Values are the means of four replicates ± SD. “*” Represents an alpha value of P

    Article Snippet: Moreover, four endocytic inhibitors, namely, dynamin inhibitor (DI: dynasore; Sigma-Aldrich), CD44-antibody (Abcam, Cambridge, MA, USA), chlorpromazine (CPZ, Aladdin, People’s Republic of China), and 5-(N -ethyl-N -isopropyl) amiloride (EIPA) were used to impede the dynamin-dependent, CD44-mediated, and macropinocytosis pathways.

    Techniques: Luciferase, Transfection, Incubation

    DiI intensity of HLPD (HLP a D, HLP b D, HLP c D, and HLP d D) in SMMC-7721 cells incubated for 4 h in the absence (normal group) and presence of ( A ) anti-CD44 antibody (50 μg/mL) pretreated for 1 h and ( B ) dynamin inhibitor I with 0.2% DMSO in the solution (DI; dynasore, 80 μM) pretreated for 1 h. Notes: Untreated group represents the cell cultured without Q-complexes. LPD: a cationic liposome, multifunctional peptide, and DNA at optimized ratios; HLPD: H represents hyaluronic acid, L represents cationic liposome that was composed of DOTAP/DOPE at a 1:1 weight ratio, P represents peptide (P a –P d refers to the different peptide used), and D represents DNA. The means of four replicates ± standard deviation are: ns, P > 0.05; *, P > 0.05; **, P > 0.01; and ***, P > 0.001. Abbreviations: FITC, fluorescein isothiocyanate; HA, hyaluronic acid; DOTAP, 1,2-dioleoyl-3-trimethylammonium-propane; DOPE, 1,2-dioleoylsn-glycero-3-phosphatidyl-ethanolamine; ns, not significant.

    Journal: International Journal of Nanomedicine

    Article Title: Effect of inserted spacer in hepatic cell-penetrating multifunctional peptide component on the DNA intracellular delivery of quaternary complexes based on modular design

    doi: 10.2147/IJN.S115381

    Figure Lengend Snippet: DiI intensity of HLPD (HLP a D, HLP b D, HLP c D, and HLP d D) in SMMC-7721 cells incubated for 4 h in the absence (normal group) and presence of ( A ) anti-CD44 antibody (50 μg/mL) pretreated for 1 h and ( B ) dynamin inhibitor I with 0.2% DMSO in the solution (DI; dynasore, 80 μM) pretreated for 1 h. Notes: Untreated group represents the cell cultured without Q-complexes. LPD: a cationic liposome, multifunctional peptide, and DNA at optimized ratios; HLPD: H represents hyaluronic acid, L represents cationic liposome that was composed of DOTAP/DOPE at a 1:1 weight ratio, P represents peptide (P a –P d refers to the different peptide used), and D represents DNA. The means of four replicates ± standard deviation are: ns, P > 0.05; *, P > 0.05; **, P > 0.01; and ***, P > 0.001. Abbreviations: FITC, fluorescein isothiocyanate; HA, hyaluronic acid; DOTAP, 1,2-dioleoyl-3-trimethylammonium-propane; DOPE, 1,2-dioleoylsn-glycero-3-phosphatidyl-ethanolamine; ns, not significant.

    Article Snippet: Moreover, four endocytic inhibitors, namely, dynamin inhibitor (DI: dynasore; Sigma-Aldrich), CD44-antibody (Abcam, Cambridge, MA, USA), chlorpromazine (CPZ, Aladdin, People’s Republic of China), and 5-(N -ethyl-N -isopropyl) amiloride (EIPA) were used to impede the dynamin-dependent, CD44-mediated, and macropinocytosis pathways.

    Techniques: Incubation, Cell Culture, Standard Deviation