dynasore Millipore Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Millipore dynasore
    Endocytosis inhibitors cause a decrease in the level of Armadillo/β-catenin both in stimulated and unstimulated cells. (A) The level of Armadillo increased in S2R + cells that had been treated with Wingless-conditioned medium (lane 2) or SB-216763 (lane 5). This was prevented by treatment with Dyngo-4a (lanes 3 and 6) or <t>Dynasore</t> (lanes 4 and 7). Lamin, Actin and Syntaxin levels were unaffected. (B) Dyngo-4a reversibly reduced the level of Armadillo in uninduced cells. (C) Dyngo-4a reduced signalling-induced accumulation of β-catenin in RKO cells. Cells were pre-incubated with SB-216763 to activate signalling and then exposed to a mixture of SB-216763 and Dyngo-4a. The total time of treatment with either drug is indicated. As in Drosophila cells, Dyngo-4a caused a decrease in β-catenin levels in unstimulated cells. A progressive decrease can be seen after 0.5, 1 and 2 hours of treatment with Dyngo-4a (no SB-216763) in lanes 9–11. (D) The effect of Dyngo-4a and SB-216763 on the level of various components of the Wnt pathway. LRP6, GSK3β and CK1α were largely unaffected, whereas the levels of APC and Axin1 dropped markedly 30–60 minutes after treatment with Dyngo-4a. SB-216763 caused an increase in the amount of β-catenin. This correlated with a decrease in phosphorylated β-catenin (pT42/S37/S33 β-catenin; lane 3), as expected because phosphorylated β-catenin reflects the activity of the degradation complex ( Hernández et al., 2012 ). By contrast, the (mild) decrease in β-catenin caused by Dyngo-4a (lanes 7 and 8) is paralleled by a similar decrease in phosphorylated β-catenin, suggesting that Dyngo-4a impacts on the level of β-catenin through a mechanism that is independent of the destruction complex.
    Dynasore, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2237 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dynasore/product/Millipore
    Average 99 stars, based on 2237 article reviews
    Price from $9.99 to $1999.99
    dynasore - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Millipore cytochalasin d
    High levels of αS reduce phagocytosis in pMac. Phagocytosis was measured by uptake of fluorescent zymosan by pMac. ( A ) Z-projection of confocal images (scale bar = 20 µm). ( B ) representative FACS plots of zymosan uptake (black line, untreated with cytochalasin D; gray, cytochalasin D-treated). ( C ) Quantification of ( B ) (3 independent experiments). ( D ) Incubation with monomeric αS for different lengths of time, then challenge with zymosan (one line). ( E ) Incubation with different concentrations of monomeric αS (24 hr), then challenge with zymosan. Values normalized to untreated mean for each of 3 independent experiments ( C , E ). Statistical analyses, one way ANOVA with Dunnett’s multiple comparisons test. Also see Figure   S4A–C .
    Cytochalasin D, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 9195 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cytochalasin d/product/Millipore
    Average 99 stars, based on 9195 article reviews
    Price from $9.99 to $1999.99
    cytochalasin d - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Millipore chlorpromazine
    High levels of αS reduce phagocytosis in pMac. Phagocytosis was measured by uptake of fluorescent zymosan by pMac. ( A ) Z-projection of confocal images (scale bar = 20 µm). ( B ) representative FACS plots of zymosan uptake (black line, untreated with cytochalasin D; gray, cytochalasin D-treated). ( C ) Quantification of ( B ) (3 independent experiments). ( D ) Incubation with monomeric αS for different lengths of time, then challenge with zymosan (one line). ( E ) Incubation with different concentrations of monomeric αS (24 hr), then challenge with zymosan. Values normalized to untreated mean for each of 3 independent experiments ( C , E ). Statistical analyses, one way ANOVA with Dunnett’s multiple comparisons test. Also see Figure   S4A–C .
    Chlorpromazine, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 800 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/chlorpromazine/product/Millipore
    Average 99 stars, based on 800 article reviews
    Price from $9.99 to $1999.99
    chlorpromazine - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Millipore bafilomycin a1
    HCMV infection is impaired when endosomal acidification is blocked. (A) HS-578T cells were pretreated with NH 4 Cl (50 mM) at 37°C for 60 min, followed by infection for 5.5 h. RNA was extracted, and HCMV UL123 expression was monitored by RT-qPCR and normalized against GAPDH amplified in the same reaction. (B) HS-578T cells were treated with NH 4 Cl at various concentrations. HCMV infection was carried out for 4.5 h, and the cells were washed with low-acid buffer for 3 min to inactivate uninternalized virus and cultured for additional 3 days without NH 4 Cl before FACS analysis. (C) Cell viability was monitored by CytoTox-One assay using the highest dose (100 mM) of NH 4 Cl. (D and E) HS-578T cells (D) and MRC-5 cells (E) were pretreated with NH 4 Cl (50 mM), monensin (25 nM), or <t>bafilomycin</t> A1 (BFLA1) (200 nM). Representative data from 3 independent experiments for each cell type are shown ( P
    Bafilomycin A1, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 7507 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bafilomycin a1/product/Millipore
    Average 99 stars, based on 7507 article reviews
    Price from $9.99 to $1999.99
    bafilomycin a1 - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Millipore nocodazole
    EHV-1gH S440A entry into ED cells. Cells were pretreated with genistein (A), dynasore (DYN) (B), MβCD (C), EIPA, or <t>nocodazole</t> (Noc) (D) before infection with EHV-1gH S440A (MOI = 5). At 8 to 12 h after infection, monolayers were detached and the
    Nocodazole, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 15733 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nocodazole/product/Millipore
    Average 99 stars, based on 15733 article reviews
    Price from $9.99 to $1999.99
    nocodazole - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Millipore wortmannin
    Cellular requirements for IPNV infection. IPNV infection of CHSE-214 cells was carried out in the presence of inhibitors of structural and functional cell components: nocodazole (10 μM), <t>wortmannin</t> (10 μM), salirasib (50 μM), casin (10 μM), Y11 (30 μM), genistein (100 μM) gefitinib (10 μM), blebbistatin (200 μM), 3-indole propionic acid (IPA-3 25 μM), NSC23766 (200 μM), rottlerin (20 μM), or no infection. CHSE-214 cells were propagated at 70–80% confluence in 24-well plates with round glass coverslips. IPNV was inoculated at an MOI of 1 for 1 h in the presence of the respective inhibitor. After 12 h of incubation at 20 °C, cells were processed by indirect immunofluorescence (IFI). Imaging of fixed slides was performed with an Olympus Spinning Disk IX81 microscope, and 250 cells were counted at each condition. Number of VP2/VP3-expressing cells is shown as a percentage, normalized to mock cells.
    Wortmannin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 9936 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/wortmannin/product/Millipore
    Average 99 stars, based on 9936 article reviews
    Price from $9.99 to $1999.99
    wortmannin - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Millipore mβcd
    Exosome internalization is dynamin and cholesterol dependent. (A) Schematic representation of the roles of dynamin-2 and cholesterol in various endocytic pathways. (B and C) Confocal images (B) and flow cytometry analysis (C) of exosome and transferrin internalization by HepG2 cells treated with dynasore. Scale bars: 10 µm. MFI (right) is normalized to that of dimethyl sulfoxide (DMSO)-treated cells. (D) Flow cytometry analysis of exosome internalization by HepG2 cells transfected with the EGFP-Dyn2K44A mutant. HepG2 cells transfected with the EGFP-tagged dominant negative Dyn2K44A mutant were incubated with PKH26-labeled exosomes. Transfected cells (EGFP + ) are gated, and the uptake of exosomes among transfected cells (EGFP + PKH26 + ) is analyzed and presented by histogram graph (left) and MFI (right). MFI is normalized to vector-transfected controls. (E to H) Confocal images (E) and flow cytometry analysis (F to H) of exosome internalization by HepG2 cells treated with cholesterol inhibitors <t>(MβCD,</t> nystatin, and filipin). Scale bars: 10 µm. For flow cytometry analysis, MFIs (right) are normalized to DMSO-treated cells. The error bars indicate the SD. *, P
    Mβcd, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1182 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mβcd/product/Millipore
    Average 99 stars, based on 1182 article reviews
    Price from $9.99 to $1999.99
    mβcd - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Millipore filipin iii
    Cholesterol depletion sensitizes cells to complement-mediated lysis but protects from SLO-mediated lysis. K562 cells were pretreated with the indicated doses of MβCD ( A , C , D ) for 15 min or <t>filipin-III</t> ( B ) for 30 min at 37 °C. For cholesterol
    Filipin Iii, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 928 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/filipin iii/product/Millipore
    Average 99 stars, based on 928 article reviews
    Price from $9.99 to $1999.99
    filipin iii - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Millipore ammonium chloride
    Cholesterol depletion sensitizes cells to complement-mediated lysis but protects from SLO-mediated lysis. K562 cells were pretreated with the indicated doses of MβCD ( A , C , D ) for 15 min or <t>filipin-III</t> ( B ) for 30 min at 37 °C. For cholesterol
    Ammonium Chloride, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2946 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ammonium chloride/product/Millipore
    Average 99 stars, based on 2946 article reviews
    Price from $9.99 to $1999.99
    ammonium chloride - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Millipore nystatin
    Cholesterol depletion sensitizes cells to complement-mediated lysis but protects from SLO-mediated lysis. K562 cells were pretreated with the indicated doses of MβCD ( A , C , D ) for 15 min or <t>filipin-III</t> ( B ) for 30 min at 37 °C. For cholesterol
    Nystatin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1775 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nystatin/product/Millipore
    Average 99 stars, based on 1775 article reviews
    Price from $9.99 to $1999.99
    nystatin - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Millipore amiloride
    Disruption of clathrin-mediated endocytosis of S15-APTs with different inhibitors Right: A549 cells were pre-incubated with: ( A ) 5 μM cytochalasin D for 30 min to block endocytosis of S15-APT QDs; ( B ) 80 μM Dynasore for 30 min; ( C ) 1 mM <t>Amiloride</t> for 10 min; ( D ) 1 μg/ml Filipin for 30 min; and ( E ) drug-free medium; followed by a further incubation of 2 h with 100 nM S15-APT QDs. Nuclei were stained with Hoechst 33342. Fluorescence microscopy was performed using an inverted confocal microscope (Zeiss LSM 710) at ×630 magnification. Left: Mean fluorescence intensity (M.F.I) values of S15-APT QDs in A549 cells incubated with different inhibitors were determined with IMARIS software for analysis of image data. The red fluorescence channel was defined between 10-100 for all presented images.
    Amiloride, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1860 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/amiloride/product/Millipore
    Average 99 stars, based on 1860 article reviews
    Price from $9.99 to $1999.99
    amiloride - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Millipore chloroquine
    Disruption of clathrin-mediated endocytosis of S15-APTs with different inhibitors Right: A549 cells were pre-incubated with: ( A ) 5 μM cytochalasin D for 30 min to block endocytosis of S15-APT QDs; ( B ) 80 μM Dynasore for 30 min; ( C ) 1 mM <t>Amiloride</t> for 10 min; ( D ) 1 μg/ml Filipin for 30 min; and ( E ) drug-free medium; followed by a further incubation of 2 h with 100 nM S15-APT QDs. Nuclei were stained with Hoechst 33342. Fluorescence microscopy was performed using an inverted confocal microscope (Zeiss LSM 710) at ×630 magnification. Left: Mean fluorescence intensity (M.F.I) values of S15-APT QDs in A549 cells incubated with different inhibitors were determined with IMARIS software for analysis of image data. The red fluorescence channel was defined between 10-100 for all presented images.
    Chloroquine, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 6123 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/chloroquine/product/Millipore
    Average 99 stars, based on 6123 article reviews
    Price from $9.99 to $1999.99
    chloroquine - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Millipore brefeldin a
    Sub-cellular localization of SLC7A5 in  Salmonella -infected cells. ( A ) HeLa cells were transfected overnight with an expression vector encoding for Myc-SLC7A5 and analyzed by IF using antibodies against Myc and Golgin-97. ( B ) HeLa cells were infected with  Salmonella  in the absence or presence of the Golgi dissassembly promoting drug Brefeldin A, added 30 min after HeLa cells were infected with  Salmonella , in order to avoid potential side-effects on bacterial entry. Next, cells were fixed and analyzed by IF using antibodies against mTOR and LAMP2. ( C ) HeLa cells infected with  Salmonella  WT for 1 h or 4 h were analyzed by IF using antibodies against the Golgi marker protein Golgin-97 and SLC7A5 ( D ) HeLa cells were infected with  Salmonella  in the absence or presence of the inhibitor of clathrin-dependent endocytosis, Dynasore, added 30 min after HeLa cells were infected with  Salmonella , in order to avoid potential side-effects on bacterial entry. Next, cells were fixed and analyzed by IF using antibodies against mTOR and LAMP2.
    Brefeldin A, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 33643 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/brefeldin a/product/Millipore
    Average 99 stars, based on 33643 article reviews
    Price from $9.99 to $1999.99
    brefeldin a - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Millipore dmso
    Dynamin plays a role in productive infection by reovirus virions and ISVPs. (A and B) L929 cells were pretreated with vehicle <t>(DMSO)</t> or <t>dynasore</t> and infected with reovirus T1L virions (A) or ISVPs (B) at an MOI of 3. After adsorption, fresh medium with
    Dmso, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 55350 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dmso/product/Millipore
    Average 99 stars, based on 55350 article reviews
    Price from $9.99 to $1999.99
    dmso - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Millipore latrunculin a
    Internalisation of mitochondria occurs by endocytosis in HOS cells, with a macropinocytic activator EGF upregulating the process. ( A – F ) Internalisation of EGFP-labelled mitochondria in HOS cells, as quantified by FACS, in the presence and absence of the endocytic inhibitors: chlorpromazine (CPZ) (100 μm) ( A ), MβCD (5 mM) ( B ), dynasore (120 μm) ( C ), EIPA (50 μm) ( D ), wortmannin (300 nM) ( E ) and <t>latrunculin</t> A (0.5 µM) ( F ). ( G , H ) EGF upregulates mitochondrial internalisation. Internalisation of EGFP-labelled mitochondria by HOS cells, as quantified by FACS, in the presence and absence of 50 nM and 100 nM EGF ( G ), 50 nM FGF and 100 nM FGF ( H ). The Y axis shows the percentage of green fluorescent cells normalised to the numbers of green cells in a control sample (cells incubated with only EGFP-labelled mitochondria for 90 min). The number of green fluorescent cells in this control sample was taken to represent 100%. The green fluorescence in all samples for inhibitor assays of mitochondrial internalisation were normalised to levels in their respective control samples. Data shown as mean values +/− s.e.m. n = 3 ( A – D and F – H ) n = 6 ( E ). *p
    Latrunculin A, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2467 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/latrunculin a/product/Millipore
    Average 99 stars, based on 2467 article reviews
    Price from $9.99 to $1999.99
    latrunculin a - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Millipore jasplakinolide
    Clathrin-mediated endocytosis and late endosome-to-lysosome trafficking is required for MHV fusion. A ) Fusion assay upon siRNA-mediated gene silencing. Three different siRNAs per gene were transfected individually into HeLa-mCC1a-ΔM15. 72 h post transfection, cells were pre-loaded with FDG by hypotonic shock. MHV-αN was allowed to bind to the cells on ice at MOI = 20 for 90 min. 100 min post warming to 37°C, cells were collected and analyzed by FACS. Fusion was determined relative to the number of FIC-positive cells observed upon mock treatment of infected cells (UNTR). Error bars represent SEM, n = 3. B ) Fusion of MHV upon treatment of cells with different inhibitors was studied as in A. Cells were pretreated with ammonium chloride (NH4Cl), Bafilomycin A1 (BafA1), Chloroquine (Chloq), Chlorpromazine (Chlopro), Monensin (Mon), Dynasore, Dyngo-4A, EIPA, Latrunculin A, (LatA), <t>Jasplakinolide</t> (Jasp), Cytochalasin B (CytoB), Cytochalasin D (DytoD), Nocodazole (Noc), U18666A, MG132, Brefelding A (BrefA), as well as with the solvents dimethyl sulfoxide (DMSO) and methanol (MeOH), protein synthesis inhibitor cyclohexamide (CHX), and MHV fusion inhibitor HR2 peptide (HR2) for 30 min at 37°C. The inhibitors were kept present during binding of MHV-αN to cells and during warming to 37°C cells for 100 min. Fusion was determined relative to the number of FIC-positive cells after mock treatment (UNTR). Error bars represent SEM, n = 3.
    Jasplakinolide, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1126 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/jasplakinolide/product/Millipore
    Average 99 stars, based on 1126 article reviews
    Price from $9.99 to $1999.99
    jasplakinolide - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Millipore mg132
    CBL-B ubiquitinates SYK. ( a ) In vitro ubiquitination of active SYK by recombinant CBL-B, CBL-B 29-483 , or C373A CBL-B 29-483 . Assays analyzed by Western blotting for SYK (upper panel) or ubiquitin (lower panel). Note, that CBL-B forms non-substrate attached poly-ubiquitin chains. * denotes unspecific bands. ( b ) In vitro ubiquitination of inactive SYK (SYK-GST) or active SYK by CBL-B 29-483 . Assays were analyzed as in ( a ). ( c ) Lysates from BM-DC stimulated with C. albicans immunoprecipitated with an anti-CBL-B antibody or IgG control. Precipitates were probed for CBL-B and SYK. Aliquots were blotted for CLB-B and SYK as a loading control. ( d ) BM-DC were stimulated with C. albicans in the presence or absence of <t>MG132</t> (10 μM) and analyzed by Western blotting. ( e ) Sequence of the TKB-binding-peptide. Antennapedia derived, and SYK derived sequences are indicated. Tyrosine 317 is shown in red, p symbolizes phosphotyrosine 317 . ( f ) In vitro ubiquitination of SYK by CBL-B 29-483 in the presence of 100 μM TKB-binding peptide. Assays analyzed by Western blotting as in ( a ). ( g ) BM-DC stimulated with C. albicans in the presence of 100 μM TKB-binding peptide and monitored for ROS production. ( P values calculated with two-way ANOVA). ( h ) Killing capacity of BM-DC as assessed by co-culture with C. albicans in the presence of 100 μM TKB-binding peptide. Data shown as means ± standard deviation. P values were calculated with Student’s t-test . For all panels, 1 representative of 3 independent repeats is shown ** P
    Mg132, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 20851 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mg132/product/Millipore
    Average 99 stars, based on 20851 article reviews
    Price from $9.99 to $1999.99
    mg132 - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Millipore monodansylcadaverine
    Effects of endocytosis inhibitors on PNP (amidine-modified and carboxylate-modified) flux across the RAECM. PNP flux was measured in the presence and absence of methyl-β-cyclodextrin (caveolin-mediated endocytosis inhibitor; Panel A ( n = 11–13)), chlorpromazine or <t>dansylcadaverine</t> (clathrin-mediated endocytosis inhibitors; Panel B ( n = 7–12)), latrunculin B or cyctochalasin D (phagocytosis and macropinocytosis inhibitors; Panel C ( n = 8–9)), and dynasore (dynamin inhibitor; Panel C ( n = 5)). FITC–cholera toxin B (CTB) (50 μg/ml apical concentration; Panel A ( n = 5)) and Alexa 594–transferrin (500 μg/ml apical concentration; Panel B ( n = 6)) flux across RAECMs was measured as positive control for caveolin- or clathrin-mediated endocytosis, respectively. *Significantly less than control; † significantly greater than control.
    Monodansylcadaverine, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 497 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monodansylcadaverine/product/Millipore
    Average 99 stars, based on 497 article reviews
    Price from $9.99 to $1999.99
    monodansylcadaverine - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Millipore blebbistatin
    P2X 7 R mediates LL-37 internalization by human macrophages. ( A and B ) dTHP-1 cells were pretreated with P2X 7 R inhibitors KN-62 (1 μM) or oxATP (100 μM) for 1 h and incubated with FAM-labeled LL-37 for an additional hour. After washing with 1× PBS three times, MFI of the cells was measured by flow cytometry ( n = 3). (A) A representative FACS plot. ( C and D ) Control (transfected with nontarget shRNA) and P2X 7 R-KD (transfected with human P2X 7 R-targeted shRNA) dTHP-1 cells were incubated with 10 μg/ml of FAM–LL-37 for 1 h. After washing with PBS three times, MFI of the cells was analyzed by flow cytometry ( n = 5). (C) One representative FACS plot. ( E ) WT, control, and P2X 7 R-KD THP-1 cells were pretreated with P2X 7 R inhibitor KN-62 (1 μM) for 1 h before incubation with FAM-labeled LL-37 for an additional hour. After washing with 1× PBS three times, MFI of the cells was measured by flow cytometry ( n = 3). ( F ) dTHP-1 cells were treated with FAM-labeled LL-37 at 37°C for 1 h. The colocalization of LL-37 and P2X 7 R was visualized using a confocal microscope. ( G ) Control and P2X 7 R-KD dTHP-1 cells were pretreated with nystatin (10 μg/ml) or dynasore (20 μM), respectively, at 37°C for 1 h. After washing with PBS three times, MFI of the cells was measured by flow cytometry ( n = 5). ( H ) dTHP-1 cells were pretreated with BzATP (100 μM, specific agonist of P2X 7 R), <t>(-)-blebbistatin</t> (200 μM, inhibitor of nonmuscle myosin II), 10 PanX (1 μM, peptide antagonist of Panx-1), FIPI (1 μM, inhibitor of PLD isoforms), wortmannin (1 μM, specific inhibitor of PI 3 K), geldanamycin (1 μM, inhibitor of hsp90), U0126 (1 μM, inhibitor of ERK MAPK), or SB203580 (1 μM, inhibitor of p38 MAPK), followed by incubation with FAM–LL-37 for 1 h. After washing with PBS three times, MFI of the cells was measured by flow cytometry ( n = 3). ( I ) The colocalization of LL-37/P2X 7 R complex and caveolin-1 or clathrin was visualized using a confocal microscope. Images in (I a ) and (I b ) are enlargements of the boxed areas. A total of 10 μg/ml of LL-37 was used in all treatments, and the confocal images are representative of at least three experiments. Scale bars, 10 μm. * p
    Blebbistatin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3436 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/blebbistatin/product/Millipore
    Average 99 stars, based on 3436 article reviews
    Price from $9.99 to $1999.99
    blebbistatin - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Millipore monensin
    FAF1 positively controls exosome number. a SH-SY5Y cells were transfected with VC or 3xFlag-FAF1 plasmid. At 24 h after transfection, the culture medium was replaced with serum-free medium containing DMSO, GW4869 (10 μM), or <t>monensin</t> (10 μM), and the cells were cultured for 24 h. Left panel: CL and concentrated CM were analyzed by western blotting with the indicated antibodies. Right panel: The graph shows the results of densitometric analysis of FAF1, Alix, and Hsc70 immunoblots in the CM shown in the left panel ( n = 3). All lanes were loaded with the same amount of total protein. b Cells were transfected with siRNA against parkin or siRNA-resistant parkin construct. At 24 h after transfection, the culture medium was replaced with serum-free medium, and the cells were cultured for 24 h. CL and concentrated CM were analyzed by western blotting with the indicated antibodies. Left panel: Representative western blots. Right panel: The graphs show the results of densitometric analysis of FAF1, Alix, and CD63 in CM, FAF1 and parkin in CL normalized to β-actin ( n = 3). c Cells plated on 150 mm dishes were transfected with VC or 3xFlag-FAF1 plasmid. At 24 h after transfection, the culture medium was replaced with exosome-depleted medium, and the cells were cultured for 48 h. Then, exosomes were isolated from the CM with ExoQuick-TC. Left panel: Distribution profile of vesicles isolated by ExoQuick-TC. The blue line indicates exosomes from VC-transfected cells, and the red line indicates exosomes from 3xFlag-FAF1-transfected cells. Right panel: Exosome numbers were normalized to the final cell number ( n = 3). The data are expressed as the mean ± S.D. of three independent experiments. Statistical comparisons were performed using ANOVA followed by Tukey’s HSD post hoc analysis (a and b) and Student’s t-test (c) . * P
    Monensin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 6165 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monensin/product/Millipore
    Average 99 stars, based on 6165 article reviews
    Price from $9.99 to $1999.99
    monensin - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Millipore eipa
    Effects of clathrin-mediated endocytic inhibitors on the entry of CHIKV into C6/36 cells. C6/36 cells were pre-treated with different inhibitors for 3 hours before CHIKV infection. Supernatants were harvested 24 hours p.i for viral plaque assays. The log virus titre is plotted against the concentrations of drug used. Dose-dependent inhibition of CHIKV entry into (a) monodansylcadaverine-, (b) chlorpromazine- and (c) dynasore-treated cells is observed. In contrast, minimal inhibition of CHIKV infectious entry into (d) filipin- and (e) nystatin-treated cells is noted. Cholesterol-dependent endocytosis of CHIKV into C6/36 cells is further analysed. Dose-dependent inhibition of CHIKV infection is observed with (f) <t>methyl-β</t> cyclodextrin treatment of C6/36 cells. Furthermore, minimal inhibition on the infectious entry of CHIKV into (g) <t>EIPA-treated</t> C6/36 cells is observed. Cell viability upon drug treatments is represented by the line graphs. The asterisk indicates * p values
    Eipa, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 395 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/eipa/product/Millipore
    Average 99 stars, based on 395 article reviews
    Price from $9.99 to $1999.99
    eipa - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    96
    Millipore ipa 3
    Cellular requirements for IPNV infection. IPNV infection of CHSE-214 cells was carried out in the presence of inhibitors of structural and functional cell components: nocodazole (10 μM), wortmannin (10 μM), salirasib (50 μM), casin (10 μM), Y11 (30 μM), genistein (100 μM) gefitinib (10 μM), blebbistatin (200 μM), 3-indole propionic acid <t>(IPA-3</t> 25 μM), NSC23766 (200 μM), rottlerin (20 μM), or no infection. CHSE-214 cells were propagated at 70–80% confluence in 24-well plates with round glass coverslips. IPNV was inoculated at an MOI of 1 for 1 h in the presence of the respective inhibitor. After 12 h of incubation at 20 °C, cells were processed by indirect immunofluorescence (IFI). Imaging of fixed slides was performed with an Olympus Spinning Disk IX81 microscope, and 250 cells were counted at each condition. Number of VP2/VP3-expressing cells is shown as a percentage, normalized to mock cells.
    Ipa 3, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 256 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ipa 3/product/Millipore
    Average 96 stars, based on 256 article reviews
    Price from $9.99 to $1999.99
    ipa 3 - by Bioz Stars, 2020-09
    96/100 stars
      Buy from Supplier

    Image Search Results


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

    Journal: Journal of Cell Science

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

    doi: 10.1242/jcs.155424

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

    Article Snippet: Endocytosis inhibition S2R+ or RKO cells were treated with Dynasore (200 µM, Sigma-Aldrich D7693), Dyngo-4a (100 µM, Abcam ab120689), or DMSO as a control, for 30 minutes before stimulation with Wingless- or Wnt-3A-conditioned medium for the indicated times.

    Techniques: Incubation, Activity Assay

    T-cad overexpression induces VE-cadherin internalization via clathrin-dependent pathway in HUVEC ells. a Confocal high-resolution images of T-cad, si-T-cad, or control HUVEC double-immunostained with antibodies against clathrin ( green fluorescence ) and VE-cadherin ( red fluorescence ). Nuclei appear blue after DAPI staining. Yellow fluorescence corresponds to VE-cadherin and clathrin co-localization ( white arrows , also in the lower panel). b To inhibit clathrin-dependent endocytosis, control, T-cad, and si-T-cad cells were treated with 80 μM dynasore for 2 h prior to staining. Cells were double-stained with anti-clathrin ( green fluorescence ) and anti-VE-cadherin ( red fluorescence ) antibodies. Nuclei were counterstained with DAPI. Lower panels demonstrate the absence of clathrin and VE-cadherin co-localization in the cytoplasm of endothelial cells. Images were captured using high-resolution confocal microscope with equal gain and offset settings. Bar 20 μm. c and d Quantification of co-localization of VE-cadherin with clathrin. Co-localization was acquired by immunofluorescence confocal analysis of representative images of four independent experiments in three triplicates and quantified using the ImageJ co-localization plug-in. Values are displayed as mean ± SEM ( n = 12; *, ** P at least

    Journal: Molecular and Cellular Biochemistry

    Article Title: Novel mechanism regulating endothelial permeability via T-cadherin-dependent VE-cadherin phosphorylation and clathrin-mediated endocytosis

    doi: 10.1007/s11010-013-1867-4

    Figure Lengend Snippet: T-cad overexpression induces VE-cadherin internalization via clathrin-dependent pathway in HUVEC ells. a Confocal high-resolution images of T-cad, si-T-cad, or control HUVEC double-immunostained with antibodies against clathrin ( green fluorescence ) and VE-cadherin ( red fluorescence ). Nuclei appear blue after DAPI staining. Yellow fluorescence corresponds to VE-cadherin and clathrin co-localization ( white arrows , also in the lower panel). b To inhibit clathrin-dependent endocytosis, control, T-cad, and si-T-cad cells were treated with 80 μM dynasore for 2 h prior to staining. Cells were double-stained with anti-clathrin ( green fluorescence ) and anti-VE-cadherin ( red fluorescence ) antibodies. Nuclei were counterstained with DAPI. Lower panels demonstrate the absence of clathrin and VE-cadherin co-localization in the cytoplasm of endothelial cells. Images were captured using high-resolution confocal microscope with equal gain and offset settings. Bar 20 μm. c and d Quantification of co-localization of VE-cadherin with clathrin. Co-localization was acquired by immunofluorescence confocal analysis of representative images of four independent experiments in three triplicates and quantified using the ImageJ co-localization plug-in. Values are displayed as mean ± SEM ( n = 12; *, ** P at least

    Article Snippet: In internalization assay HUVEC were preincubated with dynasore hydrate (Sigma) in final concentration 80 μM for 2 h, or LysoTracker® Red (Invitrogen, Ex/Em: 577/590 nm) for 60 and 120 min, or Y27632 in final concentration 10 μM (Sigma) for 12 h before immunofluorescent staining.

    Techniques: Over Expression, Fluorescence, Staining, Microscopy, Immunofluorescence

    High levels of αS reduce phagocytosis in pMac. Phagocytosis was measured by uptake of fluorescent zymosan by pMac. ( A ) Z-projection of confocal images (scale bar = 20 µm). ( B ) representative FACS plots of zymosan uptake (black line, untreated with cytochalasin D; gray, cytochalasin D-treated). ( C ) Quantification of ( B ) (3 independent experiments). ( D ) Incubation with monomeric αS for different lengths of time, then challenge with zymosan (one line). ( E ) Incubation with different concentrations of monomeric αS (24 hr), then challenge with zymosan. Values normalized to untreated mean for each of 3 independent experiments ( C , E ). Statistical analyses, one way ANOVA with Dunnett’s multiple comparisons test. Also see Figure   S4A–C .

    Journal: Scientific Reports

    Article Title: Excess α-synuclein compromises phagocytosis in iPSC-derived macrophages

    doi: 10.1038/s41598-017-09362-3

    Figure Lengend Snippet: High levels of αS reduce phagocytosis in pMac. Phagocytosis was measured by uptake of fluorescent zymosan by pMac. ( A ) Z-projection of confocal images (scale bar = 20 µm). ( B ) representative FACS plots of zymosan uptake (black line, untreated with cytochalasin D; gray, cytochalasin D-treated). ( C ) Quantification of ( B ) (3 independent experiments). ( D ) Incubation with monomeric αS for different lengths of time, then challenge with zymosan (one line). ( E ) Incubation with different concentrations of monomeric αS (24 hr), then challenge with zymosan. Values normalized to untreated mean for each of 3 independent experiments ( C , E ). Statistical analyses, one way ANOVA with Dunnett’s multiple comparisons test. Also see Figure  S4A–C .

    Article Snippet: Before and during αS stimulation pMac were treated with Cytochalasin D (Sigma, 10 µM, to inhibit actin polymerisation) or Dynasore (Cayman Chemical, 80 µM, inhibits dynamin).

    Techniques: FACS, Incubation

    HCMV infection is impaired when endosomal acidification is blocked. (A) HS-578T cells were pretreated with NH 4 Cl (50 mM) at 37°C for 60 min, followed by infection for 5.5 h. RNA was extracted, and HCMV UL123 expression was monitored by RT-qPCR and normalized against GAPDH amplified in the same reaction. (B) HS-578T cells were treated with NH 4 Cl at various concentrations. HCMV infection was carried out for 4.5 h, and the cells were washed with low-acid buffer for 3 min to inactivate uninternalized virus and cultured for additional 3 days without NH 4 Cl before FACS analysis. (C) Cell viability was monitored by CytoTox-One assay using the highest dose (100 mM) of NH 4 Cl. (D and E) HS-578T cells (D) and MRC-5 cells (E) were pretreated with NH 4 Cl (50 mM), monensin (25 nM), or bafilomycin A1 (BFLA1) (200 nM). Representative data from 3 independent experiments for each cell type are shown ( P

    Journal: Journal of Virology

    Article Title: Cell Surface THY-1 Contributes to Human Cytomegalovirus Entry via a Macropinocytosis-Like Process

    doi: 10.1128/JVI.01092-16

    Figure Lengend Snippet: HCMV infection is impaired when endosomal acidification is blocked. (A) HS-578T cells were pretreated with NH 4 Cl (50 mM) at 37°C for 60 min, followed by infection for 5.5 h. RNA was extracted, and HCMV UL123 expression was monitored by RT-qPCR and normalized against GAPDH amplified in the same reaction. (B) HS-578T cells were treated with NH 4 Cl at various concentrations. HCMV infection was carried out for 4.5 h, and the cells were washed with low-acid buffer for 3 min to inactivate uninternalized virus and cultured for additional 3 days without NH 4 Cl before FACS analysis. (C) Cell viability was monitored by CytoTox-One assay using the highest dose (100 mM) of NH 4 Cl. (D and E) HS-578T cells (D) and MRC-5 cells (E) were pretreated with NH 4 Cl (50 mM), monensin (25 nM), or bafilomycin A1 (BFLA1) (200 nM). Representative data from 3 independent experiments for each cell type are shown ( P

    Article Snippet: IPA-3 (EMD, Chicago, IL), dynasore monohydrate, and filipin III (Santa Cruz, Santa Cruz, CA), jasplakinolide (Calbiochem, San Diego, CA), 5-( N -ethyl- N -isopropyl)amiloride (EIPA), bafilomycin A1, and monensin (Sigma-Aldrich, St. Louis, MO), and (Cell Signaling Technology, Boston, MA) were used to inhibit virus entry.

    Techniques: Infection, Expressing, Quantitative RT-PCR, Amplification, Cell Culture, FACS

    EHV-1gH S440A entry into ED cells. Cells were pretreated with genistein (A), dynasore (DYN) (B), MβCD (C), EIPA, or nocodazole (Noc) (D) before infection with EHV-1gH S440A (MOI = 5). At 8 to 12 h after infection, monolayers were detached and the

    Journal: Journal of Virology

    Article Title: Glycoprotein H and ?4?1 Integrins Determine the Entry Pathway of Alphaherpesviruses

    doi: 10.1128/JVI.03522-12

    Figure Lengend Snippet: EHV-1gH S440A entry into ED cells. Cells were pretreated with genistein (A), dynasore (DYN) (B), MβCD (C), EIPA, or nocodazole (Noc) (D) before infection with EHV-1gH S440A (MOI = 5). At 8 to 12 h after infection, monolayers were detached and the

    Article Snippet: The drug concentrations used were 2 μM bafilomycin A (BFLA; Sigma) dissolved in dimethyl sulfoxide (DMSO), 10 to 100 μg/ml genistein (Sigma) dissolved in DMSO, 10 μg/ml chlorpromazine (Sigma) in PBS, 5 μg/ml filipin (Sigma) in DMSO, 5 to 20 mM methyl-β-cyclodextrin (MβCD; Sigma) in PBS, 30 μM nocodazole (Sigma) in DMSO, 75 μM 5-( N -ethyl- N -isopropyl)amiloride (EIPA; Sigma) in ethanol, and 10 to 80 μM dynasore (Sigma) in DMSO.

    Techniques: Infection

    Cellular requirements for IPNV infection. IPNV infection of CHSE-214 cells was carried out in the presence of inhibitors of structural and functional cell components: nocodazole (10 μM), wortmannin (10 μM), salirasib (50 μM), casin (10 μM), Y11 (30 μM), genistein (100 μM) gefitinib (10 μM), blebbistatin (200 μM), 3-indole propionic acid (IPA-3 25 μM), NSC23766 (200 μM), rottlerin (20 μM), or no infection. CHSE-214 cells were propagated at 70–80% confluence in 24-well plates with round glass coverslips. IPNV was inoculated at an MOI of 1 for 1 h in the presence of the respective inhibitor. After 12 h of incubation at 20 °C, cells were processed by indirect immunofluorescence (IFI). Imaging of fixed slides was performed with an Olympus Spinning Disk IX81 microscope, and 250 cells were counted at each condition. Number of VP2/VP3-expressing cells is shown as a percentage, normalized to mock cells.

    Journal: Scientific Reports

    Article Title: Infectious pancreatic necrosis virus enters CHSE-214 cells via macropinocytosis

    doi: 10.1038/s41598-017-03036-w

    Figure Lengend Snippet: Cellular requirements for IPNV infection. IPNV infection of CHSE-214 cells was carried out in the presence of inhibitors of structural and functional cell components: nocodazole (10 μM), wortmannin (10 μM), salirasib (50 μM), casin (10 μM), Y11 (30 μM), genistein (100 μM) gefitinib (10 μM), blebbistatin (200 μM), 3-indole propionic acid (IPA-3 25 μM), NSC23766 (200 μM), rottlerin (20 μM), or no infection. CHSE-214 cells were propagated at 70–80% confluence in 24-well plates with round glass coverslips. IPNV was inoculated at an MOI of 1 for 1 h in the presence of the respective inhibitor. After 12 h of incubation at 20 °C, cells were processed by indirect immunofluorescence (IFI). Imaging of fixed slides was performed with an Olympus Spinning Disk IX81 microscope, and 250 cells were counted at each condition. Number of VP2/VP3-expressing cells is shown as a percentage, normalized to mock cells.

    Article Snippet: Reagents 5-(N-Ethyl-N-isopropyl) amiloride (EIPA), blebbistatin, casin, chlorpromazine, cytochalasin D, dynasore, filipin complex, gefitinib (Iressa), genistein, IPA-3, nocodazole, NSC23766, rottlerin, salirasib, wortmannin, Y11, FluoromountTM , polyethylene glycol 8000 and Sepharose® 6B were from Sigma-Aldrich (St. Louis, MO, USA).

    Techniques: Infection, Functional Assay, Indirect Immunoperoxidase Assay, Incubation, Immunofluorescence, Imaging, Microscopy, Expressing

    Exosome internalization is dynamin and cholesterol dependent. (A) Schematic representation of the roles of dynamin-2 and cholesterol in various endocytic pathways. (B and C) Confocal images (B) and flow cytometry analysis (C) of exosome and transferrin internalization by HepG2 cells treated with dynasore. Scale bars: 10 µm. MFI (right) is normalized to that of dimethyl sulfoxide (DMSO)-treated cells. (D) Flow cytometry analysis of exosome internalization by HepG2 cells transfected with the EGFP-Dyn2K44A mutant. HepG2 cells transfected with the EGFP-tagged dominant negative Dyn2K44A mutant were incubated with PKH26-labeled exosomes. Transfected cells (EGFP + ) are gated, and the uptake of exosomes among transfected cells (EGFP + PKH26 + ) is analyzed and presented by histogram graph (left) and MFI (right). MFI is normalized to vector-transfected controls. (E to H) Confocal images (E) and flow cytometry analysis (F to H) of exosome internalization by HepG2 cells treated with cholesterol inhibitors (MβCD, nystatin, and filipin). Scale bars: 10 µm. For flow cytometry analysis, MFIs (right) are normalized to DMSO-treated cells. The error bars indicate the SD. *, P

    Journal: Journal of Virology

    Article Title: Exosomes Exploit the Virus Entry Machinery and Pathway To Transmit Alpha Interferon-Induced Antiviral Activity

    doi: 10.1128/JVI.01578-18

    Figure Lengend Snippet: Exosome internalization is dynamin and cholesterol dependent. (A) Schematic representation of the roles of dynamin-2 and cholesterol in various endocytic pathways. (B and C) Confocal images (B) and flow cytometry analysis (C) of exosome and transferrin internalization by HepG2 cells treated with dynasore. Scale bars: 10 µm. MFI (right) is normalized to that of dimethyl sulfoxide (DMSO)-treated cells. (D) Flow cytometry analysis of exosome internalization by HepG2 cells transfected with the EGFP-Dyn2K44A mutant. HepG2 cells transfected with the EGFP-tagged dominant negative Dyn2K44A mutant were incubated with PKH26-labeled exosomes. Transfected cells (EGFP + ) are gated, and the uptake of exosomes among transfected cells (EGFP + PKH26 + ) is analyzed and presented by histogram graph (left) and MFI (right). MFI is normalized to vector-transfected controls. (E to H) Confocal images (E) and flow cytometry analysis (F to H) of exosome internalization by HepG2 cells treated with cholesterol inhibitors (MβCD, nystatin, and filipin). Scale bars: 10 µm. For flow cytometry analysis, MFIs (right) are normalized to DMSO-treated cells. The error bars indicate the SD. *, P

    Article Snippet: Chemical inhibitors, including dynasore (D7693), MβCD (C4555), EIPA (A3085), IPA-3 (I2285), and rottlerin (R5648), were from Sigma-Aldrich.

    Techniques: Flow Cytometry, Cytometry, Transfection, Mutagenesis, Dominant Negative Mutation, Incubation, Labeling, Plasmid Preparation

    Effect of endocytosis inhibition on the VEGF-A-regulated cleavage of Flt1. A–D : HEK293 cells ( A and B ) transfected with hemagglutinin (HA)-Flt1-Myc-Flag (Flt1) or FLT1ΔCTD (deleted COOH-terminal domain, ΔCTD) and AG1-G1-FLT1 ( C and D ) were incubated with and without 100 ng/ml VEGF-A for 24 h with dynasore (80 μM) or methyl-β-cyclodextrin (MβCD; 50 μg/ml) for 5 h and then immunoblotted for HA, tubulin, or AF321 (Flt1) antibodies. Full-length Flt1 (FL) and the NH 2 -terminal fragment (NTF) are seen in lysates and conditioned media (CM), respectively. Neither dynasore nor MβCD have an impact on VEGF-A mediated inhibition of FLT1 and FLT1ΔCTD cleavage. A representative immunoblot in seen in A and C , and the dynasore experiments were repeated, and the pooled NTF data quantified by densitometry is shown in panel B and D . Means ± SD, n = 3. * P

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: VEGF-A selectively inhibits FLT1 ectodomain shedding independent of receptor activation and receptor endocytosis

    doi: 10.1152/ajpcell.00247.2017

    Figure Lengend Snippet: Effect of endocytosis inhibition on the VEGF-A-regulated cleavage of Flt1. A–D : HEK293 cells ( A and B ) transfected with hemagglutinin (HA)-Flt1-Myc-Flag (Flt1) or FLT1ΔCTD (deleted COOH-terminal domain, ΔCTD) and AG1-G1-FLT1 ( C and D ) were incubated with and without 100 ng/ml VEGF-A for 24 h with dynasore (80 μM) or methyl-β-cyclodextrin (MβCD; 50 μg/ml) for 5 h and then immunoblotted for HA, tubulin, or AF321 (Flt1) antibodies. Full-length Flt1 (FL) and the NH 2 -terminal fragment (NTF) are seen in lysates and conditioned media (CM), respectively. Neither dynasore nor MβCD have an impact on VEGF-A mediated inhibition of FLT1 and FLT1ΔCTD cleavage. A representative immunoblot in seen in A and C , and the dynasore experiments were repeated, and the pooled NTF data quantified by densitometry is shown in panel B and D . Means ± SD, n = 3. * P

    Article Snippet: Bafilomycin A1, GF109203X hydrochloride, genistein, monodansylcadaverine (MDC), dynasore hydrate, methyl-β-cyclodextrin (MβCD), and N-acetylleucylleucylnorleucinal (ALLN) were purchased from Sigma-Aldrich (St. Louis, MO), and dl -dithiothreitol (DTT) was from EMD Millipore (Billerica, MA).

    Techniques: Inhibition, Transfection, Incubation

    Cholesterol depletion sensitizes cells to complement-mediated lysis but protects from SLO-mediated lysis. K562 cells were pretreated with the indicated doses of MβCD ( A , C , D ) for 15 min or filipin-III ( B ) for 30 min at 37 °C. For cholesterol

    Journal: The Journal of Biological Chemistry

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

    doi: 10.1074/jbc.M111.333039

    Figure Lengend Snippet: Cholesterol depletion sensitizes cells to complement-mediated lysis but protects from SLO-mediated lysis. K562 cells were pretreated with the indicated doses of MβCD ( A , C , D ) for 15 min or filipin-III ( B ) for 30 min at 37 °C. For cholesterol

    Article Snippet: Methyl-β-cyclodextrin (MβCD), cholesterol, Filipin III, Dynasore, streptolysin O (SLO), Hepes, BSA and HBSS were purchased from Sigma (Rehovot, Israel).

    Techniques: Lysis

    Disruption of clathrin-mediated endocytosis of S15-APTs with different inhibitors Right: A549 cells were pre-incubated with: ( A ) 5 μM cytochalasin D for 30 min to block endocytosis of S15-APT QDs; ( B ) 80 μM Dynasore for 30 min; ( C ) 1 mM Amiloride for 10 min; ( D ) 1 μg/ml Filipin for 30 min; and ( E ) drug-free medium; followed by a further incubation of 2 h with 100 nM S15-APT QDs. Nuclei were stained with Hoechst 33342. Fluorescence microscopy was performed using an inverted confocal microscope (Zeiss LSM 710) at ×630 magnification. Left: Mean fluorescence intensity (M.F.I) values of S15-APT QDs in A549 cells incubated with different inhibitors were determined with IMARIS software for analysis of image data. The red fluorescence channel was defined between 10-100 for all presented images.

    Journal: Oncotarget

    Article Title: Cancer cell-selective, clathrin-mediated endocytosis of aptamer decorated nanoparticles

    doi: 10.18632/oncotarget.24772

    Figure Lengend Snippet: Disruption of clathrin-mediated endocytosis of S15-APTs with different inhibitors Right: A549 cells were pre-incubated with: ( A ) 5 μM cytochalasin D for 30 min to block endocytosis of S15-APT QDs; ( B ) 80 μM Dynasore for 30 min; ( C ) 1 mM Amiloride for 10 min; ( D ) 1 μg/ml Filipin for 30 min; and ( E ) drug-free medium; followed by a further incubation of 2 h with 100 nM S15-APT QDs. Nuclei were stained with Hoechst 33342. Fluorescence microscopy was performed using an inverted confocal microscope (Zeiss LSM 710) at ×630 magnification. Left: Mean fluorescence intensity (M.F.I) values of S15-APT QDs in A549 cells incubated with different inhibitors were determined with IMARIS software for analysis of image data. The red fluorescence channel was defined between 10-100 for all presented images.

    Article Snippet: Cytochalasin D, Filipin III, Dynasore, Amiloride, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), Hoechst 33342, and sodium azide were from Sigma-Aldrich Ltd (Rehovot, Israel).

    Techniques: Incubation, Blocking Assay, Staining, Fluorescence, Microscopy, Software

    Sub-cellular localization of SLC7A5 in  Salmonella -infected cells. ( A ) HeLa cells were transfected overnight with an expression vector encoding for Myc-SLC7A5 and analyzed by IF using antibodies against Myc and Golgin-97. ( B ) HeLa cells were infected with  Salmonella  in the absence or presence of the Golgi dissassembly promoting drug Brefeldin A, added 30 min after HeLa cells were infected with  Salmonella , in order to avoid potential side-effects on bacterial entry. Next, cells were fixed and analyzed by IF using antibodies against mTOR and LAMP2. ( C ) HeLa cells infected with  Salmonella  WT for 1 h or 4 h were analyzed by IF using antibodies against the Golgi marker protein Golgin-97 and SLC7A5 ( D ) HeLa cells were infected with  Salmonella  in the absence or presence of the inhibitor of clathrin-dependent endocytosis, Dynasore, added 30 min after HeLa cells were infected with  Salmonella , in order to avoid potential side-effects on bacterial entry. Next, cells were fixed and analyzed by IF using antibodies against mTOR and LAMP2.

    Journal: Biology Open

    Article Title: The bacterial and cellular determinants controlling the recruitment of mTOR to the Salmonella-containing vacuole

    doi: 10.1242/bio.20122840

    Figure Lengend Snippet: Sub-cellular localization of SLC7A5 in Salmonella -infected cells. ( A ) HeLa cells were transfected overnight with an expression vector encoding for Myc-SLC7A5 and analyzed by IF using antibodies against Myc and Golgin-97. ( B ) HeLa cells were infected with Salmonella in the absence or presence of the Golgi dissassembly promoting drug Brefeldin A, added 30 min after HeLa cells were infected with Salmonella , in order to avoid potential side-effects on bacterial entry. Next, cells were fixed and analyzed by IF using antibodies against mTOR and LAMP2. ( C ) HeLa cells infected with Salmonella WT for 1 h or 4 h were analyzed by IF using antibodies against the Golgi marker protein Golgin-97 and SLC7A5 ( D ) HeLa cells were infected with Salmonella in the absence or presence of the inhibitor of clathrin-dependent endocytosis, Dynasore, added 30 min after HeLa cells were infected with Salmonella , in order to avoid potential side-effects on bacterial entry. Next, cells were fixed and analyzed by IF using antibodies against mTOR and LAMP2.

    Article Snippet: 4′, 6-diamidino-2-phenylindole (DAPI), Vector Laboratories; Dynasore (no. D7693), Brefeldin A (no. B7651), D-Phenylalanine (no. P1751), L-Isoleucine (no. I2752) and were from Sigma.

    Techniques: Infection, Transfection, Expressing, Plasmid Preparation, Marker

    Dynamin plays a role in productive infection by reovirus virions and ISVPs. (A and B) L929 cells were pretreated with vehicle (DMSO) or dynasore and infected with reovirus T1L virions (A) or ISVPs (B) at an MOI of 3. After adsorption, fresh medium with

    Journal: Journal of Virology

    Article Title: Reovirus Uses Multiple Endocytic Pathways for Cell Entry

    doi: 10.1128/JVI.01861-12

    Figure Lengend Snippet: Dynamin plays a role in productive infection by reovirus virions and ISVPs. (A and B) L929 cells were pretreated with vehicle (DMSO) or dynasore and infected with reovirus T1L virions (A) or ISVPs (B) at an MOI of 3. After adsorption, fresh medium with

    Article Snippet: Cells were treated with 100 μM dynasore in dimethylsulfoxide (DMSO) (Sigma), 200 μM genistein in DMSO (Sigma), or 5 mM methyl-β-cyclodextrin (MβCD) in water (Sigma) for 1.5 h and then infected with reovirus T1L virions or ISVPs at an MOI of 3.

    Techniques: Infection, Adsorption

    Dynasore inhibits pathway-specific ligand uptake but not reovirus binding or cell viability. (A) L929 cells in suspension culture were pretreated with vehicle (DMSO) or dynasore and adsorbed with reovirus virions at a concentration of 1 × 10 5

    Journal: Journal of Virology

    Article Title: Reovirus Uses Multiple Endocytic Pathways for Cell Entry

    doi: 10.1128/JVI.01861-12

    Figure Lengend Snippet: Dynasore inhibits pathway-specific ligand uptake but not reovirus binding or cell viability. (A) L929 cells in suspension culture were pretreated with vehicle (DMSO) or dynasore and adsorbed with reovirus virions at a concentration of 1 × 10 5

    Article Snippet: Cells were treated with 100 μM dynasore in dimethylsulfoxide (DMSO) (Sigma), 200 μM genistein in DMSO (Sigma), or 5 mM methyl-β-cyclodextrin (MβCD) in water (Sigma) for 1.5 h and then infected with reovirus T1L virions or ISVPs at an MOI of 3.

    Techniques: Binding Assay, Concentration Assay

    Reovirus virions and ISVPs undergo dynamin-mediated endocytosis. Reovirus T1L virions or ISVPs were adsorbed at a concentration of 1 × 10 5 particles/cell to adherent A549 cells that had been pretreated with vehicle (DMSO) or dynasore. The monolayers

    Journal: Journal of Virology

    Article Title: Reovirus Uses Multiple Endocytic Pathways for Cell Entry

    doi: 10.1128/JVI.01861-12

    Figure Lengend Snippet: Reovirus virions and ISVPs undergo dynamin-mediated endocytosis. Reovirus T1L virions or ISVPs were adsorbed at a concentration of 1 × 10 5 particles/cell to adherent A549 cells that had been pretreated with vehicle (DMSO) or dynasore. The monolayers

    Article Snippet: Cells were treated with 100 μM dynasore in dimethylsulfoxide (DMSO) (Sigma), 200 μM genistein in DMSO (Sigma), or 5 mM methyl-β-cyclodextrin (MβCD) in water (Sigma) for 1.5 h and then infected with reovirus T1L virions or ISVPs at an MOI of 3.

    Techniques: Concentration Assay

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

    Journal: Scientific Reports

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

    doi: 10.1038/s41598-017-13227-0

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

    Article Snippet: Materials Chlorpromazine, MβCD, EIPA, latrunculin A, wortmannin, dynasore, FITC-dextran, FITC-CTB and Alexafluor 488 Transferrin were obtained from Sigma Aldrich (St. Louis, MO).

    Techniques: FACS, Incubation, Fluorescence

    Clathrin-mediated endocytosis and late endosome-to-lysosome trafficking is required for MHV fusion. A ) Fusion assay upon siRNA-mediated gene silencing. Three different siRNAs per gene were transfected individually into HeLa-mCC1a-ΔM15. 72 h post transfection, cells were pre-loaded with FDG by hypotonic shock. MHV-αN was allowed to bind to the cells on ice at MOI = 20 for 90 min. 100 min post warming to 37°C, cells were collected and analyzed by FACS. Fusion was determined relative to the number of FIC-positive cells observed upon mock treatment of infected cells (UNTR). Error bars represent SEM, n = 3. B ) Fusion of MHV upon treatment of cells with different inhibitors was studied as in A. Cells were pretreated with ammonium chloride (NH4Cl), Bafilomycin A1 (BafA1), Chloroquine (Chloq), Chlorpromazine (Chlopro), Monensin (Mon), Dynasore, Dyngo-4A, EIPA, Latrunculin A, (LatA), Jasplakinolide (Jasp), Cytochalasin B (CytoB), Cytochalasin D (DytoD), Nocodazole (Noc), U18666A, MG132, Brefelding A (BrefA), as well as with the solvents dimethyl sulfoxide (DMSO) and methanol (MeOH), protein synthesis inhibitor cyclohexamide (CHX), and MHV fusion inhibitor HR2 peptide (HR2) for 30 min at 37°C. The inhibitors were kept present during binding of MHV-αN to cells and during warming to 37°C cells for 100 min. Fusion was determined relative to the number of FIC-positive cells after mock treatment (UNTR). Error bars represent SEM, n = 3.

    Journal: PLoS Pathogens

    Article Title: Coronavirus Cell Entry Occurs through the Endo-/Lysosomal Pathway in a Proteolysis-Dependent Manner

    doi: 10.1371/journal.ppat.1004502

    Figure Lengend Snippet: Clathrin-mediated endocytosis and late endosome-to-lysosome trafficking is required for MHV fusion. A ) Fusion assay upon siRNA-mediated gene silencing. Three different siRNAs per gene were transfected individually into HeLa-mCC1a-ΔM15. 72 h post transfection, cells were pre-loaded with FDG by hypotonic shock. MHV-αN was allowed to bind to the cells on ice at MOI = 20 for 90 min. 100 min post warming to 37°C, cells were collected and analyzed by FACS. Fusion was determined relative to the number of FIC-positive cells observed upon mock treatment of infected cells (UNTR). Error bars represent SEM, n = 3. B ) Fusion of MHV upon treatment of cells with different inhibitors was studied as in A. Cells were pretreated with ammonium chloride (NH4Cl), Bafilomycin A1 (BafA1), Chloroquine (Chloq), Chlorpromazine (Chlopro), Monensin (Mon), Dynasore, Dyngo-4A, EIPA, Latrunculin A, (LatA), Jasplakinolide (Jasp), Cytochalasin B (CytoB), Cytochalasin D (DytoD), Nocodazole (Noc), U18666A, MG132, Brefelding A (BrefA), as well as with the solvents dimethyl sulfoxide (DMSO) and methanol (MeOH), protein synthesis inhibitor cyclohexamide (CHX), and MHV fusion inhibitor HR2 peptide (HR2) for 30 min at 37°C. The inhibitors were kept present during binding of MHV-αN to cells and during warming to 37°C cells for 100 min. Fusion was determined relative to the number of FIC-positive cells after mock treatment (UNTR). Error bars represent SEM, n = 3.

    Article Snippet: Stocks of 700 mM cycloheximide (CHX, Sigma), 125 µM Bafilomycin A1 (BafA1, Enzo Life Sciences), 140 mM Chloroquine (Chloq, Sigma), 120 mM Dynasore (Dyn, Enzo Life Sciences), 15 mM Dyngo-4a (Dyngo, Abcam), 100 mM Ethylisopropyl amiloride (EIPA, Enzo Life Sciences), 1 mM Nocodazole (Noc, Sigma), 1 mM Latrunculin A (LatA, Enzo Life Sciences), 2 mM Jasplakinolide (Jasp, Sigma), 20 mM Cytochalasin B (CytoB, Sigma), 20 mM Cytochalasin D (CytoD, Sigma), 25 mM MG132 (Sigma), 1 mM Brefeldin A (BrefA, Sigma), and 10 mM Furin Inhibitor I (FI, Calbiochem) were prepared in DMSO and diluted 1∶1000 in the experiments, except when indicated otherwise.

    Techniques: Single Vesicle Fusion Assay, Transfection, FACS, Infection, Binding Assay

    Endocytosis affecting agents indicate clathrin-mediated endocytosis and endosome maturation to be important in MHV infection. HeLa-mCC1a cells, inoculated with MHV-EGFPM at MOI = 0.5, were treated with the different inhibitors from 30 min prior to 8 h post inoculation (0–8 h) or from 2–8 h post inoculation (2–8 h; hatched bars): ammonium chloride (NH 4 Cl), Bafilomycin A1 (BafA1), Chloroquine (Chloq), Chlorpromazine (Chlopro), Monensin (Mon), Dynasore, Dyngo-4A, EIPA, Latrunculin A (LatA), Jasplakinolide (Jasp), Cytochalasin B (CytoB), Cytochalasin D (DytoD), Nocodazole (Noc), MG132, Brefeldin A (BrefA), as well as solvents dimethyl sulfoxide (DMSO) and methanol (MeOH). Infection was determined by FACS and displayed relative to the infection level observed in mock-treated cells (UNTR). Error bars represent SEM, n = 3.

    Journal: PLoS Pathogens

    Article Title: Coronavirus Cell Entry Occurs through the Endo-/Lysosomal Pathway in a Proteolysis-Dependent Manner

    doi: 10.1371/journal.ppat.1004502

    Figure Lengend Snippet: Endocytosis affecting agents indicate clathrin-mediated endocytosis and endosome maturation to be important in MHV infection. HeLa-mCC1a cells, inoculated with MHV-EGFPM at MOI = 0.5, were treated with the different inhibitors from 30 min prior to 8 h post inoculation (0–8 h) or from 2–8 h post inoculation (2–8 h; hatched bars): ammonium chloride (NH 4 Cl), Bafilomycin A1 (BafA1), Chloroquine (Chloq), Chlorpromazine (Chlopro), Monensin (Mon), Dynasore, Dyngo-4A, EIPA, Latrunculin A (LatA), Jasplakinolide (Jasp), Cytochalasin B (CytoB), Cytochalasin D (DytoD), Nocodazole (Noc), MG132, Brefeldin A (BrefA), as well as solvents dimethyl sulfoxide (DMSO) and methanol (MeOH). Infection was determined by FACS and displayed relative to the infection level observed in mock-treated cells (UNTR). Error bars represent SEM, n = 3.

    Article Snippet: Stocks of 700 mM cycloheximide (CHX, Sigma), 125 µM Bafilomycin A1 (BafA1, Enzo Life Sciences), 140 mM Chloroquine (Chloq, Sigma), 120 mM Dynasore (Dyn, Enzo Life Sciences), 15 mM Dyngo-4a (Dyngo, Abcam), 100 mM Ethylisopropyl amiloride (EIPA, Enzo Life Sciences), 1 mM Nocodazole (Noc, Sigma), 1 mM Latrunculin A (LatA, Enzo Life Sciences), 2 mM Jasplakinolide (Jasp, Sigma), 20 mM Cytochalasin B (CytoB, Sigma), 20 mM Cytochalasin D (CytoD, Sigma), 25 mM MG132 (Sigma), 1 mM Brefeldin A (BrefA, Sigma), and 10 mM Furin Inhibitor I (FI, Calbiochem) were prepared in DMSO and diluted 1∶1000 in the experiments, except when indicated otherwise.

    Techniques: Infection, FACS

    CBL-B ubiquitinates SYK. ( a ) In vitro ubiquitination of active SYK by recombinant CBL-B, CBL-B 29-483 , or C373A CBL-B 29-483 . Assays analyzed by Western blotting for SYK (upper panel) or ubiquitin (lower panel). Note, that CBL-B forms non-substrate attached poly-ubiquitin chains. * denotes unspecific bands. ( b ) In vitro ubiquitination of inactive SYK (SYK-GST) or active SYK by CBL-B 29-483 . Assays were analyzed as in ( a ). ( c ) Lysates from BM-DC stimulated with C. albicans immunoprecipitated with an anti-CBL-B antibody or IgG control. Precipitates were probed for CBL-B and SYK. Aliquots were blotted for CLB-B and SYK as a loading control. ( d ) BM-DC were stimulated with C. albicans in the presence or absence of MG132 (10 μM) and analyzed by Western blotting. ( e ) Sequence of the TKB-binding-peptide. Antennapedia derived, and SYK derived sequences are indicated. Tyrosine 317 is shown in red, p symbolizes phosphotyrosine 317 . ( f ) In vitro ubiquitination of SYK by CBL-B 29-483 in the presence of 100 μM TKB-binding peptide. Assays analyzed by Western blotting as in ( a ). ( g ) BM-DC stimulated with C. albicans in the presence of 100 μM TKB-binding peptide and monitored for ROS production. ( P values calculated with two-way ANOVA). ( h ) Killing capacity of BM-DC as assessed by co-culture with C. albicans in the presence of 100 μM TKB-binding peptide. Data shown as means ± standard deviation. P values were calculated with Student’s t-test . For all panels, 1 representative of 3 independent repeats is shown ** P

    Journal: Nature medicine

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

    doi: 10.1038/nm.4134

    Figure Lengend Snippet: CBL-B ubiquitinates SYK. ( a ) In vitro ubiquitination of active SYK by recombinant CBL-B, CBL-B 29-483 , or C373A CBL-B 29-483 . Assays analyzed by Western blotting for SYK (upper panel) or ubiquitin (lower panel). Note, that CBL-B forms non-substrate attached poly-ubiquitin chains. * denotes unspecific bands. ( b ) In vitro ubiquitination of inactive SYK (SYK-GST) or active SYK by CBL-B 29-483 . Assays were analyzed as in ( a ). ( c ) Lysates from BM-DC stimulated with C. albicans immunoprecipitated with an anti-CBL-B antibody or IgG control. Precipitates were probed for CBL-B and SYK. Aliquots were blotted for CLB-B and SYK as a loading control. ( d ) BM-DC were stimulated with C. albicans in the presence or absence of MG132 (10 μM) and analyzed by Western blotting. ( e ) Sequence of the TKB-binding-peptide. Antennapedia derived, and SYK derived sequences are indicated. Tyrosine 317 is shown in red, p symbolizes phosphotyrosine 317 . ( f ) In vitro ubiquitination of SYK by CBL-B 29-483 in the presence of 100 μM TKB-binding peptide. Assays analyzed by Western blotting as in ( a ). ( g ) BM-DC stimulated with C. albicans in the presence of 100 μM TKB-binding peptide and monitored for ROS production. ( P values calculated with two-way ANOVA). ( h ) Killing capacity of BM-DC as assessed by co-culture with C. albicans in the presence of 100 μM TKB-binding peptide. Data shown as means ± standard deviation. P values were calculated with Student’s t-test . For all panels, 1 representative of 3 independent repeats is shown ** P

    Article Snippet: For inhibition experiments cells were treated with the Syk inhibitor R406 (Selleckchem), anti-Dectin-1 antibody (Invivogen), MG132 (Calbiochem), or Dynasore (Sigma-Aldrich), 30min prior to stimulation (MOI: Immune cell: C. albicans = 1:2).

    Techniques: In Vitro, Recombinant, Western Blot, Immunoprecipitation, Sequencing, Binding Assay, Derivative Assay, Co-Culture Assay, Standard Deviation

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

    Journal: American Journal of Respiratory Cell and Molecular Biology

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

    doi: 10.1165/rcmb.2009-0138OC

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

    Article Snippet: Briefly, RAECMs were incubated with methyl-β-cyclodextrin (10–200 μM; Sigma), monodansylcadaverine (200 μM; Sigma), chlorpromazine (28 μM; Sigma), latrunculin B (20 μM; Sigma), cyctochalasin D (10 μM; Sigma), or dynasore (80 μM; Sigma) in both apical and basolateral fluids for 30 minutes.

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

    P2X 7 R mediates LL-37 internalization by human macrophages. ( A and B ) dTHP-1 cells were pretreated with P2X 7 R inhibitors KN-62 (1 μM) or oxATP (100 μM) for 1 h and incubated with FAM-labeled LL-37 for an additional hour. After washing with 1× PBS three times, MFI of the cells was measured by flow cytometry ( n = 3). (A) A representative FACS plot. ( C and D ) Control (transfected with nontarget shRNA) and P2X 7 R-KD (transfected with human P2X 7 R-targeted shRNA) dTHP-1 cells were incubated with 10 μg/ml of FAM–LL-37 for 1 h. After washing with PBS three times, MFI of the cells was analyzed by flow cytometry ( n = 5). (C) One representative FACS plot. ( E ) WT, control, and P2X 7 R-KD THP-1 cells were pretreated with P2X 7 R inhibitor KN-62 (1 μM) for 1 h before incubation with FAM-labeled LL-37 for an additional hour. After washing with 1× PBS three times, MFI of the cells was measured by flow cytometry ( n = 3). ( F ) dTHP-1 cells were treated with FAM-labeled LL-37 at 37°C for 1 h. The colocalization of LL-37 and P2X 7 R was visualized using a confocal microscope. ( G ) Control and P2X 7 R-KD dTHP-1 cells were pretreated with nystatin (10 μg/ml) or dynasore (20 μM), respectively, at 37°C for 1 h. After washing with PBS three times, MFI of the cells was measured by flow cytometry ( n = 5). ( H ) dTHP-1 cells were pretreated with BzATP (100 μM, specific agonist of P2X 7 R), (-)-blebbistatin (200 μM, inhibitor of nonmuscle myosin II), 10 PanX (1 μM, peptide antagonist of Panx-1), FIPI (1 μM, inhibitor of PLD isoforms), wortmannin (1 μM, specific inhibitor of PI 3 K), geldanamycin (1 μM, inhibitor of hsp90), U0126 (1 μM, inhibitor of ERK MAPK), or SB203580 (1 μM, inhibitor of p38 MAPK), followed by incubation with FAM–LL-37 for 1 h. After washing with PBS three times, MFI of the cells was measured by flow cytometry ( n = 3). ( I ) The colocalization of LL-37/P2X 7 R complex and caveolin-1 or clathrin was visualized using a confocal microscope. Images in (I a ) and (I b ) are enlargements of the boxed areas. A total of 10 μg/ml of LL-37 was used in all treatments, and the confocal images are representative of at least three experiments. Scale bars, 10 μm. * p

    Journal: The Journal of Immunology Author Choice

    Article Title: P2X7 Receptor Regulates Internalization of Antimicrobial Peptide LL-37 by Human Macrophages That Promotes Intracellular Pathogen Clearance

    doi: 10.4049/jimmunol.1402845

    Figure Lengend Snippet: P2X 7 R mediates LL-37 internalization by human macrophages. ( A and B ) dTHP-1 cells were pretreated with P2X 7 R inhibitors KN-62 (1 μM) or oxATP (100 μM) for 1 h and incubated with FAM-labeled LL-37 for an additional hour. After washing with 1× PBS three times, MFI of the cells was measured by flow cytometry ( n = 3). (A) A representative FACS plot. ( C and D ) Control (transfected with nontarget shRNA) and P2X 7 R-KD (transfected with human P2X 7 R-targeted shRNA) dTHP-1 cells were incubated with 10 μg/ml of FAM–LL-37 for 1 h. After washing with PBS three times, MFI of the cells was analyzed by flow cytometry ( n = 5). (C) One representative FACS plot. ( E ) WT, control, and P2X 7 R-KD THP-1 cells were pretreated with P2X 7 R inhibitor KN-62 (1 μM) for 1 h before incubation with FAM-labeled LL-37 for an additional hour. After washing with 1× PBS three times, MFI of the cells was measured by flow cytometry ( n = 3). ( F ) dTHP-1 cells were treated with FAM-labeled LL-37 at 37°C for 1 h. The colocalization of LL-37 and P2X 7 R was visualized using a confocal microscope. ( G ) Control and P2X 7 R-KD dTHP-1 cells were pretreated with nystatin (10 μg/ml) or dynasore (20 μM), respectively, at 37°C for 1 h. After washing with PBS three times, MFI of the cells was measured by flow cytometry ( n = 5). ( H ) dTHP-1 cells were pretreated with BzATP (100 μM, specific agonist of P2X 7 R), (-)-blebbistatin (200 μM, inhibitor of nonmuscle myosin II), 10 PanX (1 μM, peptide antagonist of Panx-1), FIPI (1 μM, inhibitor of PLD isoforms), wortmannin (1 μM, specific inhibitor of PI 3 K), geldanamycin (1 μM, inhibitor of hsp90), U0126 (1 μM, inhibitor of ERK MAPK), or SB203580 (1 μM, inhibitor of p38 MAPK), followed by incubation with FAM–LL-37 for 1 h. After washing with PBS three times, MFI of the cells was measured by flow cytometry ( n = 3). ( I ) The colocalization of LL-37/P2X 7 R complex and caveolin-1 or clathrin was visualized using a confocal microscope. Images in (I a ) and (I b ) are enlargements of the boxed areas. A total of 10 μg/ml of LL-37 was used in all treatments, and the confocal images are representative of at least three experiments. Scale bars, 10 μm. * p

    Article Snippet: PMA, 2-ME, HEPES, RPMI 1640 medium, cytochalasin B (CytoB), KN-62, oxidized ATP (oxATP), BzATP, filipin III, dynasore hydrate, nystatin, (-)-blebbistatin, wortmannin, geldanamycin, chloropromazine (CLQ), chloroquine (CLQ), FIPI hydrochloride hydrate, RIPA buffer, RPMI 1640 medium, and LPS (from Salmonella enterica ) were from Sigma-Aldrich (St. Louis, MO); U0126 and SB203580 were purchased from Tocris Bioscience (Bristol, U.K.); fluorescein-conjugated Staphylococcus aureus , FBS, and M-CSF were purchased from Life Technologies (Paisley, U.K.); Abs against clathrin, caveolin-1, and P2X7 R were from Santa Cruz Biotechnology (Santa Cruz, CA); and synthetic LL-37 (NH2 -LLGDFFRKSKEKIGKEFKRIVQRIKDFFRNLVPRTES-COOH), FAM- or TAMRA-conjugated LL-37, TAMRA-conjugated sequence-scrambled LL-37 (sLL-37;GLKLRFEFSKIKGEFLKTPEVRFRDIKLKDNRISVQR), and Panx-1 antagonist peptide 10 Panx (WRQAAFVDSY) were from Innovagen (Lund, Sweden).

    Techniques: Incubation, Labeling, Flow Cytometry, Cytometry, FACS, Transfection, shRNA, Microscopy

    FAF1 positively controls exosome number. a SH-SY5Y cells were transfected with VC or 3xFlag-FAF1 plasmid. At 24 h after transfection, the culture medium was replaced with serum-free medium containing DMSO, GW4869 (10 μM), or monensin (10 μM), and the cells were cultured for 24 h. Left panel: CL and concentrated CM were analyzed by western blotting with the indicated antibodies. Right panel: The graph shows the results of densitometric analysis of FAF1, Alix, and Hsc70 immunoblots in the CM shown in the left panel ( n = 3). All lanes were loaded with the same amount of total protein. b Cells were transfected with siRNA against parkin or siRNA-resistant parkin construct. At 24 h after transfection, the culture medium was replaced with serum-free medium, and the cells were cultured for 24 h. CL and concentrated CM were analyzed by western blotting with the indicated antibodies. Left panel: Representative western blots. Right panel: The graphs show the results of densitometric analysis of FAF1, Alix, and CD63 in CM, FAF1 and parkin in CL normalized to β-actin ( n = 3). c Cells plated on 150 mm dishes were transfected with VC or 3xFlag-FAF1 plasmid. At 24 h after transfection, the culture medium was replaced with exosome-depleted medium, and the cells were cultured for 48 h. Then, exosomes were isolated from the CM with ExoQuick-TC. Left panel: Distribution profile of vesicles isolated by ExoQuick-TC. The blue line indicates exosomes from VC-transfected cells, and the red line indicates exosomes from 3xFlag-FAF1-transfected cells. Right panel: Exosome numbers were normalized to the final cell number ( n = 3). The data are expressed as the mean ± S.D. of three independent experiments. Statistical comparisons were performed using ANOVA followed by Tukey’s HSD post hoc analysis (a and b) and Student’s t-test (c) . * P

    Journal: Cell Communication and Signaling : CCS

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

    doi: 10.1186/s12964-020-00632-8

    Figure Lengend Snippet: FAF1 positively controls exosome number. a SH-SY5Y cells were transfected with VC or 3xFlag-FAF1 plasmid. At 24 h after transfection, the culture medium was replaced with serum-free medium containing DMSO, GW4869 (10 μM), or monensin (10 μM), and the cells were cultured for 24 h. Left panel: CL and concentrated CM were analyzed by western blotting with the indicated antibodies. Right panel: The graph shows the results of densitometric analysis of FAF1, Alix, and Hsc70 immunoblots in the CM shown in the left panel ( n = 3). All lanes were loaded with the same amount of total protein. b Cells were transfected with siRNA against parkin or siRNA-resistant parkin construct. At 24 h after transfection, the culture medium was replaced with serum-free medium, and the cells were cultured for 24 h. CL and concentrated CM were analyzed by western blotting with the indicated antibodies. Left panel: Representative western blots. Right panel: The graphs show the results of densitometric analysis of FAF1, Alix, and CD63 in CM, FAF1 and parkin in CL normalized to β-actin ( n = 3). c Cells plated on 150 mm dishes were transfected with VC or 3xFlag-FAF1 plasmid. At 24 h after transfection, the culture medium was replaced with exosome-depleted medium, and the cells were cultured for 48 h. Then, exosomes were isolated from the CM with ExoQuick-TC. Left panel: Distribution profile of vesicles isolated by ExoQuick-TC. The blue line indicates exosomes from VC-transfected cells, and the red line indicates exosomes from 3xFlag-FAF1-transfected cells. Right panel: Exosome numbers were normalized to the final cell number ( n = 3). The data are expressed as the mean ± S.D. of three independent experiments. Statistical comparisons were performed using ANOVA followed by Tukey’s HSD post hoc analysis (a and b) and Student’s t-test (c) . * P

    Article Snippet: Reagents and antibodies The following reagents and antibodies used in this study were purchased commercially: TNFα from AbFrontier (Seoul, South Korea); z-IETD-fmk (caspase-8 inhibitor), mouse anti-HA antibody and rabbit anti-GM130 antibody from Abcam (Cambridge, UK); 2′,7′-dichlorofluorescin diacetate (DCFH-DA), hydrogen peroxide (H2 O2 ), 1-methyl-4-phenylpyridinium (MPP+), propidium iodide (PI), poly-D-lysine, brefeldin A (BFA), GW4869, monensin, cycloheximide (CHX), necrostatin-1 (Nec-1), DPQ, proteinase K (PK), Dynasore, heparin, Heparinase III, mouse anti-β-actin, and mouse anti-Flag antibody from Sigma-Aldrich (Saint Louis, MO, USA); 4′,6-diamidino-2-phenylindole (DAPI), horseradish peroxidase (HRP)-conjugated anti-mouse antibody, and HRP-conjugated anti-rabbit antibody from Thermo Fisher Scientific, Inc. (Rockford, IL, USA); mouse anti-Flotillin-1 from BD Biosciences (San Jose, CA, USA); bafilomycin A1, mouse anti-Alix antibody, mouse anti-β-Galactosidase antibody (40-1a), rabbit anti-Calregulin antibody, mouse anti-CD63 antibody, mouse anti-Parkin antibody, and mouse anti-FAF1 antibody from Santa Cruz Biotechnology (Dallas, TX, USA); mouse anti-Hsc70 antibody and mouse anti-Hsp90 antibody from Enzo Life Sciences (Farmingdale, NY, USA); And zVAD-fmk (z-VAD) from Calbiochem (Darmstadt, Germany).

    Techniques: Transfection, Plasmid Preparation, Cell Culture, Western Blot, Construct, Isolation

    Effects of clathrin-mediated endocytic inhibitors on the entry of CHIKV into C6/36 cells. C6/36 cells were pre-treated with different inhibitors for 3 hours before CHIKV infection. Supernatants were harvested 24 hours p.i for viral plaque assays. The log virus titre is plotted against the concentrations of drug used. Dose-dependent inhibition of CHIKV entry into (a) monodansylcadaverine-, (b) chlorpromazine- and (c) dynasore-treated cells is observed. In contrast, minimal inhibition of CHIKV infectious entry into (d) filipin- and (e) nystatin-treated cells is noted. Cholesterol-dependent endocytosis of CHIKV into C6/36 cells is further analysed. Dose-dependent inhibition of CHIKV infection is observed with (f) methyl-β cyclodextrin treatment of C6/36 cells. Furthermore, minimal inhibition on the infectious entry of CHIKV into (g) EIPA-treated C6/36 cells is observed. Cell viability upon drug treatments is represented by the line graphs. The asterisk indicates * p values

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Mosquito Cellular Factors and Functions in Mediating the Infectious entry of Chikungunya Virus

    doi: 10.1371/journal.pntd.0002050

    Figure Lengend Snippet: Effects of clathrin-mediated endocytic inhibitors on the entry of CHIKV into C6/36 cells. C6/36 cells were pre-treated with different inhibitors for 3 hours before CHIKV infection. Supernatants were harvested 24 hours p.i for viral plaque assays. The log virus titre is plotted against the concentrations of drug used. Dose-dependent inhibition of CHIKV entry into (a) monodansylcadaverine-, (b) chlorpromazine- and (c) dynasore-treated cells is observed. In contrast, minimal inhibition of CHIKV infectious entry into (d) filipin- and (e) nystatin-treated cells is noted. Cholesterol-dependent endocytosis of CHIKV into C6/36 cells is further analysed. Dose-dependent inhibition of CHIKV infection is observed with (f) methyl-β cyclodextrin treatment of C6/36 cells. Furthermore, minimal inhibition on the infectious entry of CHIKV into (g) EIPA-treated C6/36 cells is observed. Cell viability upon drug treatments is represented by the line graphs. The asterisk indicates * p values

    Article Snippet: Other inhibitors targeting alternative endocytic pathways included filipin (0.1, 0.5, 1.0, 1.5 & 2.0 µg/ml) (Sigma Aldrich) , nystatin (5, 10, 20 & 40 µM) (Sigma Aldrich) , methyl-β-cyclodextrin (2.5, 5.0, 7.5 & 10 µM) (Sigma Aldrich) and EIPA (10, 25, 50 & 100 µM) (Sigma Aldrich) , .

    Techniques: Infection, Inhibition

    Cellular requirements for IPNV infection. IPNV infection of CHSE-214 cells was carried out in the presence of inhibitors of structural and functional cell components: nocodazole (10 μM), wortmannin (10 μM), salirasib (50 μM), casin (10 μM), Y11 (30 μM), genistein (100 μM) gefitinib (10 μM), blebbistatin (200 μM), 3-indole propionic acid (IPA-3 25 μM), NSC23766 (200 μM), rottlerin (20 μM), or no infection. CHSE-214 cells were propagated at 70–80% confluence in 24-well plates with round glass coverslips. IPNV was inoculated at an MOI of 1 for 1 h in the presence of the respective inhibitor. After 12 h of incubation at 20 °C, cells were processed by indirect immunofluorescence (IFI). Imaging of fixed slides was performed with an Olympus Spinning Disk IX81 microscope, and 250 cells were counted at each condition. Number of VP2/VP3-expressing cells is shown as a percentage, normalized to mock cells.

    Journal: Scientific Reports

    Article Title: Infectious pancreatic necrosis virus enters CHSE-214 cells via macropinocytosis

    doi: 10.1038/s41598-017-03036-w

    Figure Lengend Snippet: Cellular requirements for IPNV infection. IPNV infection of CHSE-214 cells was carried out in the presence of inhibitors of structural and functional cell components: nocodazole (10 μM), wortmannin (10 μM), salirasib (50 μM), casin (10 μM), Y11 (30 μM), genistein (100 μM) gefitinib (10 μM), blebbistatin (200 μM), 3-indole propionic acid (IPA-3 25 μM), NSC23766 (200 μM), rottlerin (20 μM), or no infection. CHSE-214 cells were propagated at 70–80% confluence in 24-well plates with round glass coverslips. IPNV was inoculated at an MOI of 1 for 1 h in the presence of the respective inhibitor. After 12 h of incubation at 20 °C, cells were processed by indirect immunofluorescence (IFI). Imaging of fixed slides was performed with an Olympus Spinning Disk IX81 microscope, and 250 cells were counted at each condition. Number of VP2/VP3-expressing cells is shown as a percentage, normalized to mock cells.

    Article Snippet: Reagents 5-(N-Ethyl-N-isopropyl) amiloride (EIPA), blebbistatin, casin, chlorpromazine, cytochalasin D, dynasore, filipin complex, gefitinib (Iressa), genistein, IPA-3, nocodazole, NSC23766, rottlerin, salirasib, wortmannin, Y11, FluoromountTM , polyethylene glycol 8000 and Sepharose® 6B were from Sigma-Aldrich (St. Louis, MO, USA).

    Techniques: Infection, Functional Assay, Indirect Immunoperoxidase Assay, Incubation, Immunofluorescence, Imaging, Microscopy, Expressing