eipa Millipore Search Results


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  • 95
    Millipore anti α tubulin
    Anti α Tubulin, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 8072 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Millipore ethylisopropylamiloride eipa
    Relative cellular uptake of MPE-NPs and MPE-SeNPs in the presence of hypertonic sucrose (0.5 mol/L), chlorpromazine (25 μmol/L), simvastatin (25 μmol/L), filipin (1.0 μmol/L), <t>EIPA</t> (50 μmol/L) and latrunculin B (200 nmol/L). Paired t -test, P
    Ethylisopropylamiloride Eipa, supplied by Millipore, used in various techniques. Bioz Stars score: 89/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    75
    Millipore nhe inhibitor 5 ethylisopropyl amiloride eipa
    Relative cellular uptake of MPE-NPs and MPE-SeNPs in the presence of hypertonic sucrose (0.5 mol/L), chlorpromazine (25 μmol/L), simvastatin (25 μmol/L), filipin (1.0 μmol/L), <t>EIPA</t> (50 μmol/L) and latrunculin B (200 nmol/L). Paired t -test, P
    Nhe Inhibitor 5 Ethylisopropyl Amiloride Eipa, supplied by Millipore, used in various techniques. Bioz Stars score: 75/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore macropinocytosis inhibitor eipa
    Relative cellular uptake of MPE-NPs and MPE-SeNPs in the presence of hypertonic sucrose (0.5 mol/L), chlorpromazine (25 μmol/L), simvastatin (25 μmol/L), filipin (1.0 μmol/L), <t>EIPA</t> (50 μmol/L) and latrunculin B (200 nmol/L). Paired t -test, P
    Macropinocytosis Inhibitor Eipa, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    75
    Enzo Biochem eipa
    Relative cellular uptake of MPE-NPs and MPE-SeNPs in the presence of hypertonic sucrose (0.5 mol/L), chlorpromazine (25 μmol/L), simvastatin (25 μmol/L), filipin (1.0 μmol/L), <t>EIPA</t> (50 μmol/L) and latrunculin B (200 nmol/L). Paired t -test, P
    Eipa, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 75/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    81
    Millipore na h inhibitor eipa
    Relative cellular uptake of MPE-NPs and MPE-SeNPs in the presence of hypertonic sucrose (0.5 mol/L), chlorpromazine (25 μmol/L), simvastatin (25 μmol/L), filipin (1.0 μmol/L), <t>EIPA</t> (50 μmol/L) and latrunculin B (200 nmol/L). Paired t -test, P
    Na H Inhibitor Eipa, supplied by Millipore, used in various techniques. Bioz Stars score: 81/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    75
    Millipore eipa sigma stock solutions
    Relative cellular uptake of MPE-NPs and MPE-SeNPs in the presence of hypertonic sucrose (0.5 mol/L), chlorpromazine (25 μmol/L), simvastatin (25 μmol/L), filipin (1.0 μmol/L), <t>EIPA</t> (50 μmol/L) and latrunculin B (200 nmol/L). Paired t -test, P
    Eipa Sigma Stock Solutions, supplied by Millipore, used in various techniques. Bioz Stars score: 75/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    ApexBio 3 amino 6 chloro n diaminomethylene 5 ethyl isopropyl amino pyrazine 2 carboxamide eipa
    Relative cellular uptake of MPE-NPs and MPE-SeNPs in the presence of hypertonic sucrose (0.5 mol/L), chlorpromazine (25 μmol/L), simvastatin (25 μmol/L), filipin (1.0 μmol/L), <t>EIPA</t> (50 μmol/L) and latrunculin B (200 nmol/L). Paired t -test, P
    3 Amino 6 Chloro N Diaminomethylene 5 Ethyl Isopropyl Amino Pyrazine 2 Carboxamide Eipa, supplied by ApexBio, used in various techniques. Bioz Stars score: 86/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 86 stars, based on 4 article reviews
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    78
    Millipore erk inhibitor eip i
    Relative cellular uptake of MPE-NPs and MPE-SeNPs in the presence of hypertonic sucrose (0.5 mol/L), chlorpromazine (25 μmol/L), simvastatin (25 μmol/L), filipin (1.0 μmol/L), <t>EIPA</t> (50 μmol/L) and latrunculin B (200 nmol/L). Paired t -test, P
    Erk Inhibitor Eip I, supplied by Millipore, used in various techniques. Bioz Stars score: 78/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore amiloride
    DC-EBIO given after <t>amiloride</t> does not affect I SC . The columns show the I SC after mounting the monolayers in the Ussing chambers (baseline), I SC after addition of amiloride and I SC after adding DC-EBIO (0.3 mM). Mean currents ± SEM of 14 monolayers are shown. After blocking the ENaC with amiloride, DC-EBIO did not significantly alter the I SC (ns).
    Amiloride, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 1410 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Millipore amiloride analog ethyl
    DC-EBIO given after <t>amiloride</t> does not affect I SC . The columns show the I SC after mounting the monolayers in the Ussing chambers (baseline), I SC after addition of amiloride and I SC after adding DC-EBIO (0.3 mM). Mean currents ± SEM of 14 monolayers are shown. After blocking the ENaC with amiloride, DC-EBIO did not significantly alter the I SC (ns).
    Amiloride Analog Ethyl, supplied by Millipore, used in various techniques. Bioz Stars score: 78/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    Millipore macropinocytosis inhibitor ethyl isopropyl amiloride
    DC-EBIO given after <t>amiloride</t> does not affect I SC . The columns show the I SC after mounting the monolayers in the Ussing chambers (baseline), I SC after addition of amiloride and I SC after adding DC-EBIO (0.3 mM). Mean currents ± SEM of 14 monolayers are shown. After blocking the ENaC with amiloride, DC-EBIO did not significantly alter the I SC (ns).
    Macropinocytosis Inhibitor Ethyl Isopropyl Amiloride, supplied by Millipore, used in various techniques. Bioz Stars score: 77/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    Millipore macropinocytic inhibitor 5
    DC-EBIO given after <t>amiloride</t> does not affect I SC . The columns show the I SC after mounting the monolayers in the Ussing chambers (baseline), I SC after addition of amiloride and I SC after adding DC-EBIO (0.3 mM). Mean currents ± SEM of 14 monolayers are shown. After blocking the ENaC with amiloride, DC-EBIO did not significantly alter the I SC (ns).
    Macropinocytic Inhibitor 5, supplied by Millipore, used in various techniques. Bioz Stars score: 87/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore 5 n n dimethyl amiloride
    DC-EBIO given after <t>amiloride</t> does not affect I SC . The columns show the I SC after mounting the monolayers in the Ussing chambers (baseline), I SC after addition of amiloride and I SC after adding DC-EBIO (0.3 mM). Mean currents ± SEM of 14 monolayers are shown. After blocking the ENaC with amiloride, DC-EBIO did not significantly alter the I SC (ns).
    5 N N Dimethyl Amiloride, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Millipore na h exchange inhibitor 5
    DC-EBIO given after <t>amiloride</t> does not affect I SC . The columns show the I SC after mounting the monolayers in the Ussing chambers (baseline), I SC after addition of amiloride and I SC after adding DC-EBIO (0.3 mM). Mean currents ± SEM of 14 monolayers are shown. After blocking the ENaC with amiloride, DC-EBIO did not significantly alter the I SC (ns).
    Na H Exchange Inhibitor 5, supplied by Millipore, used in various techniques. Bioz Stars score: 78/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Millipore macropinocytosis inhibitor 5
    DC-EBIO given after <t>amiloride</t> does not affect I SC . The columns show the I SC after mounting the monolayers in the Ussing chambers (baseline), I SC after addition of amiloride and I SC after adding DC-EBIO (0.3 mM). Mean currents ± SEM of 14 monolayers are shown. After blocking the ENaC with amiloride, DC-EBIO did not significantly alter the I SC (ns).
    Macropinocytosis Inhibitor 5, supplied by Millipore, used in various techniques. Bioz Stars score: 79/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    Enzo Biochem 5 n ethyl n isopropyl amiloride
    DC-EBIO given after <t>amiloride</t> does not affect I SC . The columns show the I SC after mounting the monolayers in the Ussing chambers (baseline), I SC after addition of amiloride and I SC after adding DC-EBIO (0.3 mM). Mean currents ± SEM of 14 monolayers are shown. After blocking the ENaC with amiloride, DC-EBIO did not significantly alter the I SC (ns).
    5 N Ethyl N Isopropyl Amiloride, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 80/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    Millipore nhe1 inhibitor 5 n ethyl n isopropyl amiloride
    DC-EBIO given after <t>amiloride</t> does not affect I SC . The columns show the I SC after mounting the monolayers in the Ussing chambers (baseline), I SC after addition of amiloride and I SC after adding DC-EBIO (0.3 mM). Mean currents ± SEM of 14 monolayers are shown. After blocking the ENaC with amiloride, DC-EBIO did not significantly alter the I SC (ns).
    Nhe1 Inhibitor 5 N Ethyl N Isopropyl Amiloride, supplied by Millipore, used in various techniques. Bioz Stars score: 80/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    81
    Millipore ski
    Changes in 20-KDa myosin light chain (MLC 20 ) phosphorylation with the elevation of transmural pressure, and the effects of <t>S1P</t> or <t>SKI</t> application. Results are representative of immunoblots from five independent preparations. Results are expressed as means ± SEMs (n = 5). TMP: transmural pressure.
    Ski, supplied by Millipore, used in various techniques. Bioz Stars score: 81/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore cytochalasin d
    Macropinocytotic internalization of Ebola virions is GP-dependent. (A) Co-localization of SNX5 with VSV pseudotyped with EBOV GP. Labeled VSV particles pseudotyped with EBOV GP (DiI-VSVΔ*G-GP) or VSV G (DiI-VSVΔ*G-G) were adsorbed to eGFP-SNX5-expressing Vero cells for 30 min on ice. The cells were then incubated at 37°C and time-lapse images were acquired at 20-second intervals over a period of 30 min by using confocal laser scanning microscope. Still frames of DiI-VSVΔ*G-GP (left panel) and DiI-VSVΔ*G-G (right panel) at 10 min after the temperature shift are shown. DiI-pseudovirions that co-localize with eGFP-SNX5 are indicated by arrows. Scale bars, 10 µm. (B) Graphic representation of the co-localization of EBOV GP-pseudotyped VSV virions with Rab7-positive vesicles. Co-localization of DiI-VSVΔ*G-GP (green bars) with Rab7-positive vesicles was analyzed at the indicated time points as indicated in the Materials and Methods . Experiments were performed in triplicate and the results are presented as the mean ± standard deviation. Results obtained for DiI-EbolaΔVP30 (blue bars) and DiI-VSVΔ*G-G (red bars) are shown for comparison. (C) Effect of macropinocytosis inhibitors on the co-localization of DiI-labeled VSV pseudovirions with eGFP-Rab7-positive vesicles. Vero cells expressing eGFP-Rab7 were pretreated with <t>CytoD,</t> Wort, LY294002 or EIPA for 30 min at 37°C; control cells were treated with DMSO. DiI-labeled VSVΔ*G-GP (green bars) or VSVΔ*G-G (red bars) were adsorbed to cells for 30 min on ice. The cells were then incubated at 37°C in the presence of inhibitors for 2 h. Co-localization of DiI-pseudovirions with eGFP-Rab7-positive vesicles was analyzed as described in the Materials and Methods . Experiments were carried out in triplicate and the results are presented as the mean ± standard deviation. (D) Effect of macropinocytosis inhibitors on the infectivity of VSV pseudovirions. Vero cells were treated with individual inhibitors for 30 min at 37°C and infected with VSVΔG*-GP (green bars) or VSVΔG*-G (red bars) in the presence of the inhibitor. 1 h post-infection, surface-bound virions were removed by trypsin and the cells were cultured for 24 h in the absence of inhibitors. The infection efficiency of each pseudovirus was determined by measuring the number of GFP-positive cells using with conventional fluorescent microscope. Each experiment was performed in triplicate and the relative infection efficiencies are presented as the mean ± SD.
    Cytochalasin D, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 7585 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    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: 95/100, based on 165 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore fibroblast growth factor
    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.
    Fibroblast Growth Factor, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 742 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore blebbistatin
    Actomyosin network dynamics are required for DYNA-IND entry by IAV. (A and B) Compounds affecting actin polymerization/de-polymerization (20 µM cytochalasin D [CYTD], 20 µM cytochalasin B [CYTB], 1 µM Latrunculin A [LATR], 1 µM Jasplakinolide [JASP]), inhibitors of MLCK (5 µM ML-7 and 5 µM ML-9) and an inhibitor of myosin II, (80 µM <t>Blebbistatin</t> [BLEB]) were examined for their effect on DYNA-IND IAV entry in PBS (A) or DYNA-DEP IAV entry (in PBS, 10% FCS and 80 µM dynasor) (B) using the Gluc-entry assay (HeLa cells; strain WSN; MOI 0.5; incubation with inhibitors from 1 hr prior to infection to 2 hrs p.i.). Luciferase activity was determined 16 hrs p.i. (RLU plotted on the y-axis relative to the activity obtained in absence of compounds affecting the actomysoin network (grey bars, 100%). (C) An example of confocal images of HeLa cells, expressing a wildtype Rab5-GFP fusion protein (Rab5 wt) (upper panel, green fluorescence identifies transfected cells) or a dominant-negative mutant of a Rab5-GFP fusion protein (RAB5 DN; lower panel), inoculated with IAV (strain WSN; MOI 1). Infection was performed for 4 hrs after which cells were fixed and stained (red staining with a monoclonal antibody directed against NP identifies infected cells; In the example of panel C infection was performed in PBS in order to examine the DYNA-DEP entry pathway) Similar experiments were performed for GFP fusion proteins with dominant-negative and wildtype MLCK (MLCK DN and MLCK wt) and Myosin II-tail domain (MyoII-tail) or MyosinII-head domain (MyoII-head). Transfected cells were infected under DYNA-DEP (PBS) and DYNA-IND (PBS, 10% FCS and 80 µM dynasor) entry conditions. (D–F) Results were quantified by counting > 100 cells (experiment performed in triplo; transfected cells, infected cells and cells that were transfected as well as infected were counted). Relative infection of transfected cells was plotted (taking infection of non-transfected cells in the same sample as 100%). Thus, whereas Rab5 wt transfected cells are not significantly reduced or enhanced in infection compared to non-transfected cells (D, black bars), Rab5 DN transfected cells (D, grey bars) are infected at lower levels both under DYNA-DEP and DYNA-IND entry conditions. In contrast, cells transfected with MCLK DN (E) or MyoII-head (F) were infected at significantly lower levels via the DYNA-IND pathway while the DYNA-DEP pathway was not affected. Transfection efficiency for the different constructs was as follows: RAB5 wt (61%); RAB5 DN (45%); MLCK wt (50%); MLCK DN (49%); MyoII-tail (69%); MyoII-head (55%).
    Blebbistatin, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 2619 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    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: 95/100, based on 812 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore chlorpromazine
    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.
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    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
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    Millipore methyl β cyclodextrin mβcd
    Collagen internalization is regulated by Dynamin-2, phosphoinositide-3-kinase/AKT, and actin cytoskeleton. Human HSCs were infected with K44A mutant Dynamin-2 adenovirus for 24 h or pretreated with Dynasore ( A ), blebbistatin, ML7, or Cytochalasin D ( B ), <t>methyl-β-cyclodextrin</t> <t>(MβCD),</t> filipin, nystatin, or 0.5 M sucrose ( C ), or wortmannin or AKT inhibitor ( D ) for 30 min before DQ-collagen I (0.5 μg/ml) incubation for 3 h. Cells were subjected to FACS analysis to measure matrix protein endocytosis ( n = 4, * P
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    RSV infection induces actin rearrangement. (A). RSV (moi ∼0.5) was bound to HeLa cells at 4°C, unbound virus was removed and cells warmed to 37°C, fixed at indicated times, and stained with phalloidin-AF488 (pseudocolored white) and anti-F-AF647 (red) antibody. Images represent Z-stack projections acquired with a confocal microscope. Arrowheads show actin blebs formed at the cell surface. (B). RSV (moi ∼30) was incubated with HeLa cells for 30 or 120 min at 37°C. Samples were processed according to the kit manufacturer's protocol (Cytoskeleton Inc.). Controls included mock-treated cells and cells either treated with F actin enhancer or F actin depolymerizing agent. (left) The F and G actin fractions were resolved by SDS-PAGE and western blots probed with anti-actin antibody. (right) Quantification of actin protein bands intensities by densitometry. (C–F). HeLa cells were pretreated with solvent (MOCK), cytochalasin D (CytoD), <t>latrunculin</t> A (LatA), jasplakinolide (Jas), nocodazole (Noc), taxol (Tax), NCS23766, pirl1, IPA-3, wiskostatin (Wisko), CK-869, CT04, Y24632 at indicated concentrations and each inhibitor was continuously present during following steps of the experiment: (C, E). Cells where infected with RSV (moi ∼3) or SFV-ZsGreen (moi ∼0.5) for up to 6 hours before FACS analysis of GFP expressing cells. (D, F). RSV (moi ∼3) was bound to the cells at 4°C followed by 1 h of internalization at 37°C. Cells were trypsinized, fixed and stained with anti-N-AF488 antibody, and the MFI of AF-488 measured by FACS.
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    PHEV propagation depends upon cholesterol fluidity but does not involve caveola/raft-dependent endocytosis. (A) PHEV entry was assessed in cells treated with DMSO (control), <t>MβCD</t> (5 mM), <t>Nys/Prog</t> (30/10 μM), or genistein (100 μM) for 60 min. Following binding on ice, PHEV was allowed to internalize at 37°C for 60 min. Surface-bound virus was removed, and the cells were fixed and visualized by use of a confocal microscope. (B) Quantitative results for the average PHEV and CTB fluorescence intensities in cells pretreated with pharmacological inhibitors are presented in a histogram. (C) PHEV internalization after treatment of Neuro-2a cells with the indicated agents for 1 h was quantified by qRT-PCR. Data are shown as percentages of PHEV uptake compared to that of control-treated cells. (D) PHEV infection assays were carried out with MβCD-, Nys/Prog-, and genistein-treated cells. PHEV genome equivalents were estimated by qRT-PCR, using the 2 −ΔΔ CT method, while viral protein synthesis levels were estimated by Western blotting with anti-PHEV-S antibody. GAPDH was used as a loading control. (E) A PHEV uptake and propagation assay was carried out with siCav1-transfected Neuro-2a cells. Infected cells were lysed to quantitate viral RNA copy numbers by qRT-PCR, and the silencing efficiency of siCav1 was analyzed by Western blotting using anti-caveolin-1 antibody. (F) The lack of involvement of the caveola/lipid raft-mediated route was demonstrated by the stability of PHEV infection in siCav1-transfected cells. Pretransfected cells were incubated with PHEV for 24 h to allow virus propagation. The cells were then fixed, stained with anti-Cav1 (red) and anti-PHEV (green) antibodies, and visualized by confocal microscopy. Data shown are means ± SD for three independent experiments. *, P
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    PHEV propagation depends upon cholesterol fluidity but does not involve caveola/raft-dependent endocytosis. (A) PHEV entry was assessed in cells treated with DMSO (control), <t>MβCD</t> (5 mM), <t>Nys/Prog</t> (30/10 μM), or genistein (100 μM) for 60 min. Following binding on ice, PHEV was allowed to internalize at 37°C for 60 min. Surface-bound virus was removed, and the cells were fixed and visualized by use of a confocal microscope. (B) Quantitative results for the average PHEV and CTB fluorescence intensities in cells pretreated with pharmacological inhibitors are presented in a histogram. (C) PHEV internalization after treatment of Neuro-2a cells with the indicated agents for 1 h was quantified by qRT-PCR. Data are shown as percentages of PHEV uptake compared to that of control-treated cells. (D) PHEV infection assays were carried out with MβCD-, Nys/Prog-, and genistein-treated cells. PHEV genome equivalents were estimated by qRT-PCR, using the 2 −ΔΔ CT method, while viral protein synthesis levels were estimated by Western blotting with anti-PHEV-S antibody. GAPDH was used as a loading control. (E) A PHEV uptake and propagation assay was carried out with siCav1-transfected Neuro-2a cells. Infected cells were lysed to quantitate viral RNA copy numbers by qRT-PCR, and the silencing efficiency of siCav1 was analyzed by Western blotting using anti-caveolin-1 antibody. (F) The lack of involvement of the caveola/lipid raft-mediated route was demonstrated by the stability of PHEV infection in siCav1-transfected cells. Pretransfected cells were incubated with PHEV for 24 h to allow virus propagation. The cells were then fixed, stained with anti-Cav1 (red) and anti-PHEV (green) antibodies, and visualized by confocal microscopy. Data shown are means ± SD for three independent experiments. *, P
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    PHEV propagation depends upon cholesterol fluidity but does not involve caveola/raft-dependent endocytosis. (A) PHEV entry was assessed in cells treated with DMSO (control), <t>MβCD</t> (5 mM), <t>Nys/Prog</t> (30/10 μM), or genistein (100 μM) for 60 min. Following binding on ice, PHEV was allowed to internalize at 37°C for 60 min. Surface-bound virus was removed, and the cells were fixed and visualized by use of a confocal microscope. (B) Quantitative results for the average PHEV and CTB fluorescence intensities in cells pretreated with pharmacological inhibitors are presented in a histogram. (C) PHEV internalization after treatment of Neuro-2a cells with the indicated agents for 1 h was quantified by qRT-PCR. Data are shown as percentages of PHEV uptake compared to that of control-treated cells. (D) PHEV infection assays were carried out with MβCD-, Nys/Prog-, and genistein-treated cells. PHEV genome equivalents were estimated by qRT-PCR, using the 2 −ΔΔ CT method, while viral protein synthesis levels were estimated by Western blotting with anti-PHEV-S antibody. GAPDH was used as a loading control. (E) A PHEV uptake and propagation assay was carried out with siCav1-transfected Neuro-2a cells. Infected cells were lysed to quantitate viral RNA copy numbers by qRT-PCR, and the silencing efficiency of siCav1 was analyzed by Western blotting using anti-caveolin-1 antibody. (F) The lack of involvement of the caveola/lipid raft-mediated route was demonstrated by the stability of PHEV infection in siCav1-transfected cells. Pretransfected cells were incubated with PHEV for 24 h to allow virus propagation. The cells were then fixed, stained with anti-Cav1 (red) and anti-PHEV (green) antibodies, and visualized by confocal microscopy. Data shown are means ± SD for three independent experiments. *, P
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    Image Search Results


    Relative cellular uptake of MPE-NPs and MPE-SeNPs in the presence of hypertonic sucrose (0.5 mol/L), chlorpromazine (25 μmol/L), simvastatin (25 μmol/L), filipin (1.0 μmol/L), EIPA (50 μmol/L) and latrunculin B (200 nmol/L). Paired t -test, P

    Journal: Acta Pharmaceutica Sinica. B

    Article Title: Selenium-layered nanoparticles serving for oral delivery of phytomedicines with hypoglycemic activity to synergistically potentiate the antidiabetic effect

    doi: 10.1016/j.apsb.2018.09.009

    Figure Lengend Snippet: Relative cellular uptake of MPE-NPs and MPE-SeNPs in the presence of hypertonic sucrose (0.5 mol/L), chlorpromazine (25 μmol/L), simvastatin (25 μmol/L), filipin (1.0 μmol/L), EIPA (50 μmol/L) and latrunculin B (200 nmol/L). Paired t -test, P

    Article Snippet: Simvastatin, chlorpromazine, filipin, latrunculin B and ethylisopropylamiloride (EIPA) were purchased from Sigma–Aldrich (MO, USA).

    Techniques:

    The effects of small molecule inhibitors on the uptake of ANG by SH-SY5Y and C8-D1A cells. SH-SY5Y (A) or C8-D1A (B) cells were incubated with ANG after pre-treatment with Cytochalasin D (Cyto. D) with or without Dyngo4a for 30 minutes, Similar treatments were performed with EIPA and Nystatin on ANG uptake by SH-SY5Y (C,E) and C8-D1A (D,F) cells. Cells were also incubated with Alexa fluor 594 labelled Transferrin or Dextran, and FAM labelled TAT peptide as uptake control. Mean fluorescence levels per square micrometre were determined in order to compare uptake between treatments (G,H). Scale bar: 25 μm. Error bars SEM. The nucleus and cytoplasm of least twenty cells were analysed from each of the three independent experiments performed. The mean fluorescence was compared by ANOVA, with Dunnett’s post-hoc comparison to the untreated control at each time point. n = 3, *P

    Journal: PLoS ONE

    Article Title: The cellular uptake of angiogenin, an angiogenic and neurotrophic factor is through multiple pathways and largely dynamin independent

    doi: 10.1371/journal.pone.0193302

    Figure Lengend Snippet: The effects of small molecule inhibitors on the uptake of ANG by SH-SY5Y and C8-D1A cells. SH-SY5Y (A) or C8-D1A (B) cells were incubated with ANG after pre-treatment with Cytochalasin D (Cyto. D) with or without Dyngo4a for 30 minutes, Similar treatments were performed with EIPA and Nystatin on ANG uptake by SH-SY5Y (C,E) and C8-D1A (D,F) cells. Cells were also incubated with Alexa fluor 594 labelled Transferrin or Dextran, and FAM labelled TAT peptide as uptake control. Mean fluorescence levels per square micrometre were determined in order to compare uptake between treatments (G,H). Scale bar: 25 μm. Error bars SEM. The nucleus and cytoplasm of least twenty cells were analysed from each of the three independent experiments performed. The mean fluorescence was compared by ANOVA, with Dunnett’s post-hoc comparison to the untreated control at each time point. n = 3, *P

    Article Snippet: For effects on ANG uptake by inhibitors, treatments were performed in DMEM:F12 with 0.5% KOSR (Life Technologies) at the following inhibitor concentrations: 80μM Dynasore (Abcam), 50μM Dyngo4a (Abcam), 5 μg/mL Cytochalasin D (Sigma), 10μM EIPA (Sigma), 25μg/mL nystatin (Sigma) 18mM Brefeldin A (Sigma), 0.1mM Chloroquine (Sigma), 0.1μM Wortmannin (Sigma), 1mM Lovastatin (Sigma) and 17mM Nocodazole (Sigma).

    Techniques: Incubation, Fluorescence

    Amiloride and EIPA induced a decrease in [Na + ] i in non-capacitated sperm. Sperm from the cauda epididymus were placed in medium that did not support capacitation, loaded with CoroNa Red, washed and incubated for an additional 60 minutes in the absence

    Journal: Journal of Cell Science

    Article Title: Flow cytometry analysis reveals a decrease in intracellular sodium during sperm capacitation

    doi: 10.1242/jcs.093344

    Figure Lengend Snippet: Amiloride and EIPA induced a decrease in [Na + ] i in non-capacitated sperm. Sperm from the cauda epididymus were placed in medium that did not support capacitation, loaded with CoroNa Red, washed and incubated for an additional 60 minutes in the absence

    Article Snippet: Chemicals were obtained from the following sources: bovine serum albumin (BSA; fatty acid-free), dibutyryl-cAMP (Bt2cAMP), 3-isobutyl-1-methylxanthine (IBMX), 4-bromo-calcium ionophore A23187, amiloride hydrochloride hydrate, 5-( N -ethyl- N -isopropyl) amiloride (EIPA), sodium gluconate and carbonyl cyanide m -chlorophenylhydrazone (CCCP) were from Sigma (St Louis, MO); H-89 was from Cayman Chemical Company (Ann Arbor, MI); rabbit monoclonal anti-phosphorylated PKA substrate (clone 100G7E) was purchased from Cell Signaling (Danvers, MA); anti-phosphorylated tyrosine (Y- P ) monoclonal antibody (clone 4G10) was from Upstate Biotechnology (Lake Placid, NY); horseradish peroxidase-conjugated anti-mouse and anti-rabbit IgG were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA) and GE Life Sciences, respectively; propidium iodide and CoroNaRed sodium fluorescent probes were from Invitrogen.

    Techniques: Incubation

    MNV-1 infection is independent of phagocytosis and/or macropinocytosis. (A) RAW 264.7 cells were pretreated with 10 μM cytochalasin D (Cyto D), 200 μM amiloride (EIPA), or mock control before infection with Listeria monocytogenes strain

    Journal: Journal of Virology

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

    doi: 10.1128/JVI.00331-10

    Figure Lengend Snippet: MNV-1 infection is independent of phagocytosis and/or macropinocytosis. (A) RAW 264.7 cells were pretreated with 10 μM cytochalasin D (Cyto D), 200 μM amiloride (EIPA), or mock control before infection with Listeria monocytogenes strain

    Article Snippet: Cells were pretreated with chloroquine, neuraminidase, dynasore, chlorpromazine, sucrose, nystatin, cytochalasin D, amiloride (EIPA) (all purchased from Sigma-Aldrich, MO), or vehicle control for 30 min, or 60 min for MβCD.

    Techniques: Infection

    Effect of Na + /H + exchanger ( NHE ) and Rho kinase inhibition on ET ‐1‐induced migration of rat pulmonary arterial smooth muscle cells ( PASMC s). Plot shows effect of 10 μ mol/L ethyl‐isopropyl amiloride ( EIPA ), 1 μ mol/L dimethyl amiloride ( DMA ), or 10 μ mol/L Y‐27632 on migration of PASMC s treated for 24 h with either 10 −8 mol/L ET ‐1 or vehicle ( n = 3–19 per group). Migration was measured by Transwell assay. Bars show mean with standard error. *Indicates significant difference between groups ( P

    Journal: Physiological Reports

    Article Title: Rho kinase and Na+/H+ exchanger mediate endothelin‐1‐induced pulmonary arterial smooth muscle cell proliferation and migration. Rho kinase and Na+/H+ exchanger mediate endothelin‐1‐induced pulmonary arterial smooth muscle cell proliferation and migration

    doi: 10.14814/phy2.13698

    Figure Lengend Snippet: Effect of Na + /H + exchanger ( NHE ) and Rho kinase inhibition on ET ‐1‐induced migration of rat pulmonary arterial smooth muscle cells ( PASMC s). Plot shows effect of 10 μ mol/L ethyl‐isopropyl amiloride ( EIPA ), 1 μ mol/L dimethyl amiloride ( DMA ), or 10 μ mol/L Y‐27632 on migration of PASMC s treated for 24 h with either 10 −8 mol/L ET ‐1 or vehicle ( n = 3–19 per group). Migration was measured by Transwell assay. Bars show mean with standard error. *Indicates significant difference between groups ( P

    Article Snippet: 10 μ mol/L ethyl‐isopropyl amiloride (EIPA; Sigma) or 1 μ mol/L dimethyl amiloride (DMA; Sigma) was used to inhibit NHE.

    Techniques: Inhibition, Migration, Transwell Assay

    Effect of Na + /H + exchanger ( NHE ) and Rho kinase inhibition on ET ‐1‐induced proliferation of rat pulmonary arterial smooth muscle cells ( PASMC s). Plot shows effect of 10 μ mol/L ethyl‐isopropyl amiloride ( EIPA ), 1 μ mol/L dimethyl amiloride ( DMA ), or 10 μ mol/L Y‐27632 on proliferation of PASMC s treated for 24 h with either 10 −8 mol/L ET ‐1 or vehicle ( n = 4–17 per group). Proliferation was measured by ELISA for BrdU incorporation. Bars show mean with standard error. *Indicates significant difference between groups ( P

    Journal: Physiological Reports

    Article Title: Rho kinase and Na+/H+ exchanger mediate endothelin‐1‐induced pulmonary arterial smooth muscle cell proliferation and migration. Rho kinase and Na+/H+ exchanger mediate endothelin‐1‐induced pulmonary arterial smooth muscle cell proliferation and migration

    doi: 10.14814/phy2.13698

    Figure Lengend Snippet: Effect of Na + /H + exchanger ( NHE ) and Rho kinase inhibition on ET ‐1‐induced proliferation of rat pulmonary arterial smooth muscle cells ( PASMC s). Plot shows effect of 10 μ mol/L ethyl‐isopropyl amiloride ( EIPA ), 1 μ mol/L dimethyl amiloride ( DMA ), or 10 μ mol/L Y‐27632 on proliferation of PASMC s treated for 24 h with either 10 −8 mol/L ET ‐1 or vehicle ( n = 4–17 per group). Proliferation was measured by ELISA for BrdU incorporation. Bars show mean with standard error. *Indicates significant difference between groups ( P

    Article Snippet: 10 μ mol/L ethyl‐isopropyl amiloride (EIPA; Sigma) or 1 μ mol/L dimethyl amiloride (DMA; Sigma) was used to inhibit NHE.

    Techniques: Inhibition, Enzyme-linked Immunosorbent Assay, BrdU Incorporation Assay

    pH microdomains are sensitive to NHE inhibition with 5-( N -ethyl- N -isopropyl) amiloride (EIPA). The left kidney was loaded with BCECF, and blood pressure was measured as described in . A : low-power BCECF ratio image of a larger renal cortical area

    Journal: American Journal of Physiology - Renal Physiology

    Article Title: Local pH domains regulate NHE3-mediated Na+ reabsorption in the renal proximal tubule

    doi: 10.1152/ajprenal.00174.2014

    Figure Lengend Snippet: pH microdomains are sensitive to NHE inhibition with 5-( N -ethyl- N -isopropyl) amiloride (EIPA). The left kidney was loaded with BCECF, and blood pressure was measured as described in . A : low-power BCECF ratio image of a larger renal cortical area

    Article Snippet: Albumin (1% BSA)-containing saline and 5-( N -ethyl- N -isopropyl) amiloride (EIPA; 1.5 mg/kg, Sigma) injections were administered via a catheter inserted into the right jugular vein.

    Techniques: Inhibition

    Changes in the rate of uterine fluid secretion in the presence of different protein/enzyme inhibitors in (a) rats at different phases of the oestrous cycle and (b) steroid treated ovariectomized rats. Results indicated that fluid secretion rate was increased under E influence. Glibenclamide, DIDS and ACTZ administration caused reduction in fluid secretion rate at Es, Ps and following E treatment. Interestingly, EIPA caused a marked increase in the secretion rate under E influence. The rate of fluid secretion was decreased under P influence. ACTZ and EIPA administration caused the rate to increase at Ds and following P treatment. E: 0.2 µg 17β-oestradiol, P: 4mg progesterone, E+P: 0.2 µg 17β-oestradiol + 4mg progesterone. Ps: proestrus, Es: estrus, Ms: metestrus, Ds: diestrus. G: glibenclamide, ACTZ: acetazolamide, DIDS: 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium salt hydrate and EIPA: 5-(N-Ethyl-N-isopropyl)-amiloride † as compared to Es or control, * as compared to the group without inhibitor. (*, † p

    Journal: International Journal of Medical Sciences

    Article Title: In-Vivo Functional Study on the Involvement of CFTR, SLC26A6, NHE-1 and CA Isoenzymes II and XII in Uterine Fluid pH, Volume and Electrolyte Regulation in Rats under Different Sex-Steroid Influence

    doi: 10.7150/ijms.5918

    Figure Lengend Snippet: Changes in the rate of uterine fluid secretion in the presence of different protein/enzyme inhibitors in (a) rats at different phases of the oestrous cycle and (b) steroid treated ovariectomized rats. Results indicated that fluid secretion rate was increased under E influence. Glibenclamide, DIDS and ACTZ administration caused reduction in fluid secretion rate at Es, Ps and following E treatment. Interestingly, EIPA caused a marked increase in the secretion rate under E influence. The rate of fluid secretion was decreased under P influence. ACTZ and EIPA administration caused the rate to increase at Ds and following P treatment. E: 0.2 µg 17β-oestradiol, P: 4mg progesterone, E+P: 0.2 µg 17β-oestradiol + 4mg progesterone. Ps: proestrus, Es: estrus, Ms: metestrus, Ds: diestrus. G: glibenclamide, ACTZ: acetazolamide, DIDS: 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium salt hydrate and EIPA: 5-(N-Ethyl-N-isopropyl)-amiloride † as compared to Es or control, * as compared to the group without inhibitor. (*, † p

    Article Snippet: In order to investigate the functional involvement of the proteins of interest, the following inhibitors were dissolved into the perfusion fluid and were then perfused into the uterine horn: acetazolamide (ACTZ), (CA inhibitor) (Sigma) at 100µM , glibenclamide (CFTR inhibitor) (Sigma) at 200 µM , 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium salt hydrate (DIDS) (SLC26A6 inhibitor) (Sigma) at 500 µM and 5-(N-Ethyl-N-isopropyl)-amiloride (EIPA) (NHE inhibitor) (Sigma) at 100 µM .

    Techniques: Mass Spectrometry

    Uterine fluid Na + concentration with and without the presence of protein/enzyme inhibitors in (a) rats at different stages of the oestrous cycle and (b) steroid replaced ovariectomized rats. Na + concentration was high under E influence, however was low under P influence. Glibenclamide and DIDS administration caused a decrease in Na+ concentration at Es, Ps and following E treatment. Meanwhile, ACTZ and EIPA administration caused an increase in Na + concentration under P influence. E: 0.2µg 17β-oestradiol, P: 4mg progesterone, E+P: 0.2 µg 17β-oestradiol + 4mg progesterone. Ps: proestrus, Es: estrus, Ms: metestrus, Ds: diestrus. G: glibenclamide, ACTZ: acetazolamide, DIDS: 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium salt hydrate and EIPA: 5-(N-Ethyl-N-isopropyl)-amiloride. †as compared to control and Es, * as compared to the group without inhibitor (*, † :p

    Journal: International Journal of Medical Sciences

    Article Title: In-Vivo Functional Study on the Involvement of CFTR, SLC26A6, NHE-1 and CA Isoenzymes II and XII in Uterine Fluid pH, Volume and Electrolyte Regulation in Rats under Different Sex-Steroid Influence

    doi: 10.7150/ijms.5918

    Figure Lengend Snippet: Uterine fluid Na + concentration with and without the presence of protein/enzyme inhibitors in (a) rats at different stages of the oestrous cycle and (b) steroid replaced ovariectomized rats. Na + concentration was high under E influence, however was low under P influence. Glibenclamide and DIDS administration caused a decrease in Na+ concentration at Es, Ps and following E treatment. Meanwhile, ACTZ and EIPA administration caused an increase in Na + concentration under P influence. E: 0.2µg 17β-oestradiol, P: 4mg progesterone, E+P: 0.2 µg 17β-oestradiol + 4mg progesterone. Ps: proestrus, Es: estrus, Ms: metestrus, Ds: diestrus. G: glibenclamide, ACTZ: acetazolamide, DIDS: 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium salt hydrate and EIPA: 5-(N-Ethyl-N-isopropyl)-amiloride. †as compared to control and Es, * as compared to the group without inhibitor (*, † :p

    Article Snippet: In order to investigate the functional involvement of the proteins of interest, the following inhibitors were dissolved into the perfusion fluid and were then perfused into the uterine horn: acetazolamide (ACTZ), (CA inhibitor) (Sigma) at 100µM , glibenclamide (CFTR inhibitor) (Sigma) at 200 µM , 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium salt hydrate (DIDS) (SLC26A6 inhibitor) (Sigma) at 500 µM and 5-(N-Ethyl-N-isopropyl)-amiloride (EIPA) (NHE inhibitor) (Sigma) at 100 µM .

    Techniques: Concentration Assay, Mass Spectrometry

    Uterine fluid HCO3- concentration with and without the presence of protein/enzyme inhibitors in (a) rats at different phases of the oestrous cycle and (b) ovariectomized rats receiving steroid replacement. Uterine fluid HCO 3 - concentration was increased under E influence. Glibenclamide, DIDS and ACTZ administration caused a decrease in HCO3- concentration at Es, Ps and following E treatment. HCO 3 - concentration was reduced under P influence. ACTZ and EIPA administration caused an increase in HCO3- concentration at Ds and following P treatment. E: 0.2 µg 17β-oestradiol, P: progesterone, E+P: 0.2 µg 17β-oestradiol + 4mg progesterone. Ps: proestrus, Es: estrus, Ms: metestrus, Ds: diestrus. G: glibenclamide, ACTZ: acetazolamide DIDS: 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium salt hydrate and EIPA: 5-(N-Ethyl-N-isopropyl)-amiloride. † as compared to Es or control, * as compared to the group without inhibitor.(*, † :p

    Journal: International Journal of Medical Sciences

    Article Title: In-Vivo Functional Study on the Involvement of CFTR, SLC26A6, NHE-1 and CA Isoenzymes II and XII in Uterine Fluid pH, Volume and Electrolyte Regulation in Rats under Different Sex-Steroid Influence

    doi: 10.7150/ijms.5918

    Figure Lengend Snippet: Uterine fluid HCO3- concentration with and without the presence of protein/enzyme inhibitors in (a) rats at different phases of the oestrous cycle and (b) ovariectomized rats receiving steroid replacement. Uterine fluid HCO 3 - concentration was increased under E influence. Glibenclamide, DIDS and ACTZ administration caused a decrease in HCO3- concentration at Es, Ps and following E treatment. HCO 3 - concentration was reduced under P influence. ACTZ and EIPA administration caused an increase in HCO3- concentration at Ds and following P treatment. E: 0.2 µg 17β-oestradiol, P: progesterone, E+P: 0.2 µg 17β-oestradiol + 4mg progesterone. Ps: proestrus, Es: estrus, Ms: metestrus, Ds: diestrus. G: glibenclamide, ACTZ: acetazolamide DIDS: 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium salt hydrate and EIPA: 5-(N-Ethyl-N-isopropyl)-amiloride. † as compared to Es or control, * as compared to the group without inhibitor.(*, † :p

    Article Snippet: In order to investigate the functional involvement of the proteins of interest, the following inhibitors were dissolved into the perfusion fluid and were then perfused into the uterine horn: acetazolamide (ACTZ), (CA inhibitor) (Sigma) at 100µM , glibenclamide (CFTR inhibitor) (Sigma) at 200 µM , 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium salt hydrate (DIDS) (SLC26A6 inhibitor) (Sigma) at 500 µM and 5-(N-Ethyl-N-isopropyl)-amiloride (EIPA) (NHE inhibitor) (Sigma) at 100 µM .

    Techniques: Concentration Assay, Mass Spectrometry

    Changes in uterine fluid pH in (a) rats throughout oestrous cycle phases and (b) steroid treated ovariectomized rats. The pH in the presence of different protein channel/enzyme inhibitors was determined. Higher pH was noted at Es and Ps and following E treatment, while lower pH was noted at Ds and following P treatment. Glibenclamide, DIDS and ACTZ administration caused a decrease in pH under E influence, while ACTZ and EIPA administration caused an increase in pH under P influence. Meanwhile, in E+P group, the pH was not significantly differing from the control. EIPA caused pH increase at Ms and following E+P treatment. E: 0.2µg 17β-oestradiol, P: 4mg progesterone, E+P: 0.2µg 17β-oestradiol + 4mg progesterone. Ps: proestrus, Es: estrus, Ms: metestrus, Ds: diestrus, G: glibenclamide, ACTZ: acetazolamide, DIDS: 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium salt hydrate and EIPA: 5-(N-Ethyl-N-isopropyl)-amiloride. † as compared to Es or control, * as compared to the group without inhibitors. (*, † p

    Journal: International Journal of Medical Sciences

    Article Title: In-Vivo Functional Study on the Involvement of CFTR, SLC26A6, NHE-1 and CA Isoenzymes II and XII in Uterine Fluid pH, Volume and Electrolyte Regulation in Rats under Different Sex-Steroid Influence

    doi: 10.7150/ijms.5918

    Figure Lengend Snippet: Changes in uterine fluid pH in (a) rats throughout oestrous cycle phases and (b) steroid treated ovariectomized rats. The pH in the presence of different protein channel/enzyme inhibitors was determined. Higher pH was noted at Es and Ps and following E treatment, while lower pH was noted at Ds and following P treatment. Glibenclamide, DIDS and ACTZ administration caused a decrease in pH under E influence, while ACTZ and EIPA administration caused an increase in pH under P influence. Meanwhile, in E+P group, the pH was not significantly differing from the control. EIPA caused pH increase at Ms and following E+P treatment. E: 0.2µg 17β-oestradiol, P: 4mg progesterone, E+P: 0.2µg 17β-oestradiol + 4mg progesterone. Ps: proestrus, Es: estrus, Ms: metestrus, Ds: diestrus, G: glibenclamide, ACTZ: acetazolamide, DIDS: 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium salt hydrate and EIPA: 5-(N-Ethyl-N-isopropyl)-amiloride. † as compared to Es or control, * as compared to the group without inhibitors. (*, † p

    Article Snippet: In order to investigate the functional involvement of the proteins of interest, the following inhibitors were dissolved into the perfusion fluid and were then perfused into the uterine horn: acetazolamide (ACTZ), (CA inhibitor) (Sigma) at 100µM , glibenclamide (CFTR inhibitor) (Sigma) at 200 µM , 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium salt hydrate (DIDS) (SLC26A6 inhibitor) (Sigma) at 500 µM and 5-(N-Ethyl-N-isopropyl)-amiloride (EIPA) (NHE inhibitor) (Sigma) at 100 µM .

    Techniques: Mass Spectrometry

    Uterine fluid Cl- concentrations with and without the presence of protein/enzyme inhibitors in (a) rats at different stages of the oestrous cycle and (b) ovariectomized rats receiving steroid replacement. The highest Cl - level was observed under E influence. Glibenclamide and ACTZ administration caused a decrease in Cl- concentration in rats at Es, Ps and following E treatment. Meanwhile, Cl - concentration was reduced under P influence. ACTZ and EIPA administration caused an increase in Cl- concentration at Ds and following P treatment. E: 0.2µg 17β-oestradiol, P: 4mg progesterone, E+P: 0.2 µg 17β-oestradiol + 4mg progesterone. Ps: proestrus, Es: estrus, Ms: metestrus, Ds: diestrus. G: glibenclamide, ACTZ: acetazolamide, DIDS: 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium salt hydrate and EIPA: 5-(N-Ethyl-N-isopropyl)-amiloride † as compared to Es or control, * as compared with the group without inhibitor. (*:p

    Journal: International Journal of Medical Sciences

    Article Title: In-Vivo Functional Study on the Involvement of CFTR, SLC26A6, NHE-1 and CA Isoenzymes II and XII in Uterine Fluid pH, Volume and Electrolyte Regulation in Rats under Different Sex-Steroid Influence

    doi: 10.7150/ijms.5918

    Figure Lengend Snippet: Uterine fluid Cl- concentrations with and without the presence of protein/enzyme inhibitors in (a) rats at different stages of the oestrous cycle and (b) ovariectomized rats receiving steroid replacement. The highest Cl - level was observed under E influence. Glibenclamide and ACTZ administration caused a decrease in Cl- concentration in rats at Es, Ps and following E treatment. Meanwhile, Cl - concentration was reduced under P influence. ACTZ and EIPA administration caused an increase in Cl- concentration at Ds and following P treatment. E: 0.2µg 17β-oestradiol, P: 4mg progesterone, E+P: 0.2 µg 17β-oestradiol + 4mg progesterone. Ps: proestrus, Es: estrus, Ms: metestrus, Ds: diestrus. G: glibenclamide, ACTZ: acetazolamide, DIDS: 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium salt hydrate and EIPA: 5-(N-Ethyl-N-isopropyl)-amiloride † as compared to Es or control, * as compared with the group without inhibitor. (*:p

    Article Snippet: In order to investigate the functional involvement of the proteins of interest, the following inhibitors were dissolved into the perfusion fluid and were then perfused into the uterine horn: acetazolamide (ACTZ), (CA inhibitor) (Sigma) at 100µM , glibenclamide (CFTR inhibitor) (Sigma) at 200 µM , 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium salt hydrate (DIDS) (SLC26A6 inhibitor) (Sigma) at 500 µM and 5-(N-Ethyl-N-isopropyl)-amiloride (EIPA) (NHE inhibitor) (Sigma) at 100 µM .

    Techniques: Concentration Assay, Mass Spectrometry

    DC-EBIO given after amiloride does not affect I SC . The columns show the I SC after mounting the monolayers in the Ussing chambers (baseline), I SC after addition of amiloride and I SC after adding DC-EBIO (0.3 mM). Mean currents ± SEM of 14 monolayers are shown. After blocking the ENaC with amiloride, DC-EBIO did not significantly alter the I SC (ns).

    Journal: British Journal of Pharmacology

    Article Title: Benzimidazolones enhance the function of epithelial Na+ transport

    doi: 10.1111/bph.12027

    Figure Lengend Snippet: DC-EBIO given after amiloride does not affect I SC . The columns show the I SC after mounting the monolayers in the Ussing chambers (baseline), I SC after addition of amiloride and I SC after adding DC-EBIO (0.3 mM). Mean currents ± SEM of 14 monolayers are shown. After blocking the ENaC with amiloride, DC-EBIO did not significantly alter the I SC (ns).

    Article Snippet: Amiloride (A-7410, Sigma, Chemical Company, St. Louis, MO), amphotericin B (A-4888, Sigma), benzamil (B-2417, Sigma), CFTRinh 172 (3430, Tocris, Bristol, UK), chlorzoxazone (C-4397, Sigma), DC-EBIO (5,6-dichloro-1-ethyl-1,3-dihydro-2-benzimidazolone) (1422, Tocris), 1-EBIO (1-ethyl-1,3-dihydro-2-benzimidazolone) (SML0034, Sigma), EIPA (5-( N -Ethyl- N -isopropyl)amiloride) (A-3085, Sigma), N -methyl- d -glucamine (NMDG+ , M-2004, Sigma), ouabain (O-3125, Sigma).

    Techniques: Blocking Assay

    DC-EBIO enhances the magnitude of CFTR inh 172-sensitive currents. The figure shows the CFTR inh 172-sensitive I SC after amiloride inhibition and DC-EBIO (0.3 mM) stimulation compared with control monolayers without DC-EBIO stimulation. Mean CFTR inh 172-sensitive currents ± SEM of 23 (control) and 20 (DC-EBIO) monolayers are shown; the CFTR inh 172-sensitive I SC was much smaller than the amiloride-sensitive I SC but significantly increased by DC-EBIO treatment (** P

    Journal: British Journal of Pharmacology

    Article Title: Benzimidazolones enhance the function of epithelial Na+ transport

    doi: 10.1111/bph.12027

    Figure Lengend Snippet: DC-EBIO enhances the magnitude of CFTR inh 172-sensitive currents. The figure shows the CFTR inh 172-sensitive I SC after amiloride inhibition and DC-EBIO (0.3 mM) stimulation compared with control monolayers without DC-EBIO stimulation. Mean CFTR inh 172-sensitive currents ± SEM of 23 (control) and 20 (DC-EBIO) monolayers are shown; the CFTR inh 172-sensitive I SC was much smaller than the amiloride-sensitive I SC but significantly increased by DC-EBIO treatment (** P

    Article Snippet: Amiloride (A-7410, Sigma, Chemical Company, St. Louis, MO), amphotericin B (A-4888, Sigma), benzamil (B-2417, Sigma), CFTRinh 172 (3430, Tocris, Bristol, UK), chlorzoxazone (C-4397, Sigma), DC-EBIO (5,6-dichloro-1-ethyl-1,3-dihydro-2-benzimidazolone) (1422, Tocris), 1-EBIO (1-ethyl-1,3-dihydro-2-benzimidazolone) (SML0034, Sigma), EIPA (5-( N -Ethyl- N -isopropyl)amiloride) (A-3085, Sigma), N -methyl- d -glucamine (NMDG+ , M-2004, Sigma), ouabain (O-3125, Sigma).

    Techniques: Inhibition

    DC-EBIO enhances membrane conductance in whole cell measurements. Whole cell current measurements in A549 cells ( n = 7) with Na + as the only permeant ion on either side of the cell membrane. Addition of 10 and 100 μM DC-EBIO resulted in a concentration-dependent increase in cell membrane conductance, which was almost completely amiloride-sensitive. Currents were normalized by the membrane conductance under control conditions and shown as mean ± SEM.

    Journal: British Journal of Pharmacology

    Article Title: Benzimidazolones enhance the function of epithelial Na+ transport

    doi: 10.1111/bph.12027

    Figure Lengend Snippet: DC-EBIO enhances membrane conductance in whole cell measurements. Whole cell current measurements in A549 cells ( n = 7) with Na + as the only permeant ion on either side of the cell membrane. Addition of 10 and 100 μM DC-EBIO resulted in a concentration-dependent increase in cell membrane conductance, which was almost completely amiloride-sensitive. Currents were normalized by the membrane conductance under control conditions and shown as mean ± SEM.

    Article Snippet: Amiloride (A-7410, Sigma, Chemical Company, St. Louis, MO), amphotericin B (A-4888, Sigma), benzamil (B-2417, Sigma), CFTRinh 172 (3430, Tocris, Bristol, UK), chlorzoxazone (C-4397, Sigma), DC-EBIO (5,6-dichloro-1-ethyl-1,3-dihydro-2-benzimidazolone) (1422, Tocris), 1-EBIO (1-ethyl-1,3-dihydro-2-benzimidazolone) (SML0034, Sigma), EIPA (5-( N -Ethyl- N -isopropyl)amiloride) (A-3085, Sigma), N -methyl- d -glucamine (NMDG+ , M-2004, Sigma), ouabain (O-3125, Sigma).

    Techniques: Concentration Assay

    Benzimidazolones increase short-circuit currents. Effects of 1-EBIO (3 mM), DC-EBIO (0.3 mM) and chlorzoxazone (1 mM) on I SC of rat FDLE cell monolayers. The figure shows the increase in I SC induced by addition of one of the compounds tested (benzimidazolone-induced I SC , A), the I SC decrease after adding amiloride (10 μM, the amiloride-sensitive I SC (B) and the I SC decrease after addition of amiloride and ouabain (1 mM, ouabain-sensitive I SC (B). Mean treatment-associated changes in current ± SEM of 22 (control), 6 (1-EBIO), 16 (DC-EBIO) and 16 (chlorzoxazone) monolayers are shown and were significantly increased by the substances tested (* P

    Journal: British Journal of Pharmacology

    Article Title: Benzimidazolones enhance the function of epithelial Na+ transport

    doi: 10.1111/bph.12027

    Figure Lengend Snippet: Benzimidazolones increase short-circuit currents. Effects of 1-EBIO (3 mM), DC-EBIO (0.3 mM) and chlorzoxazone (1 mM) on I SC of rat FDLE cell monolayers. The figure shows the increase in I SC induced by addition of one of the compounds tested (benzimidazolone-induced I SC , A), the I SC decrease after adding amiloride (10 μM, the amiloride-sensitive I SC (B) and the I SC decrease after addition of amiloride and ouabain (1 mM, ouabain-sensitive I SC (B). Mean treatment-associated changes in current ± SEM of 22 (control), 6 (1-EBIO), 16 (DC-EBIO) and 16 (chlorzoxazone) monolayers are shown and were significantly increased by the substances tested (* P

    Article Snippet: Amiloride (A-7410, Sigma, Chemical Company, St. Louis, MO), amphotericin B (A-4888, Sigma), benzamil (B-2417, Sigma), CFTRinh 172 (3430, Tocris, Bristol, UK), chlorzoxazone (C-4397, Sigma), DC-EBIO (5,6-dichloro-1-ethyl-1,3-dihydro-2-benzimidazolone) (1422, Tocris), 1-EBIO (1-ethyl-1,3-dihydro-2-benzimidazolone) (SML0034, Sigma), EIPA (5-( N -Ethyl- N -isopropyl)amiloride) (A-3085, Sigma), N -methyl- d -glucamine (NMDG+ , M-2004, Sigma), ouabain (O-3125, Sigma).

    Techniques:

    I SC trace of the permeabilized basolateral membrane measurement. After addition of DC-EBIO (300 μM) amphotericin B (100 μM) was applied basolaterally. At the maximum current increase, amiloride (10 μM) was added apically to determine max. amiloride-sensitive I SC .

    Journal: British Journal of Pharmacology

    Article Title: Benzimidazolones enhance the function of epithelial Na+ transport

    doi: 10.1111/bph.12027

    Figure Lengend Snippet: I SC trace of the permeabilized basolateral membrane measurement. After addition of DC-EBIO (300 μM) amphotericin B (100 μM) was applied basolaterally. At the maximum current increase, amiloride (10 μM) was added apically to determine max. amiloride-sensitive I SC .

    Article Snippet: Amiloride (A-7410, Sigma, Chemical Company, St. Louis, MO), amphotericin B (A-4888, Sigma), benzamil (B-2417, Sigma), CFTRinh 172 (3430, Tocris, Bristol, UK), chlorzoxazone (C-4397, Sigma), DC-EBIO (5,6-dichloro-1-ethyl-1,3-dihydro-2-benzimidazolone) (1422, Tocris), 1-EBIO (1-ethyl-1,3-dihydro-2-benzimidazolone) (SML0034, Sigma), EIPA (5-( N -Ethyl- N -isopropyl)amiloride) (A-3085, Sigma), N -methyl- d -glucamine (NMDG+ , M-2004, Sigma), ouabain (O-3125, Sigma).

    Techniques:

    Benzimidazolones enhance the magnitude of the amiloride-sensitive apical conductance. The monolayers were subjected to a 145:5 apical to basolateral Na + gradient, the basolateral membrane was permeabilized by addition of 100 μM amphotericin B, resulting in an amphotericin B-induced increase in I SC (A). The maximum amiloride-sensitive I SC (amil max , B) reduction was caused by the addition of 10 μM amiloride into the apical compartment at the maximum current increase. The data of 13 (control), 5 (1-EBIO), 3 (DC-EBIO) and 12 (chlorzoxazone) monolayers are represented as mean ± SEM (** P

    Journal: British Journal of Pharmacology

    Article Title: Benzimidazolones enhance the function of epithelial Na+ transport

    doi: 10.1111/bph.12027

    Figure Lengend Snippet: Benzimidazolones enhance the magnitude of the amiloride-sensitive apical conductance. The monolayers were subjected to a 145:5 apical to basolateral Na + gradient, the basolateral membrane was permeabilized by addition of 100 μM amphotericin B, resulting in an amphotericin B-induced increase in I SC (A). The maximum amiloride-sensitive I SC (amil max , B) reduction was caused by the addition of 10 μM amiloride into the apical compartment at the maximum current increase. The data of 13 (control), 5 (1-EBIO), 3 (DC-EBIO) and 12 (chlorzoxazone) monolayers are represented as mean ± SEM (** P

    Article Snippet: Amiloride (A-7410, Sigma, Chemical Company, St. Louis, MO), amphotericin B (A-4888, Sigma), benzamil (B-2417, Sigma), CFTRinh 172 (3430, Tocris, Bristol, UK), chlorzoxazone (C-4397, Sigma), DC-EBIO (5,6-dichloro-1-ethyl-1,3-dihydro-2-benzimidazolone) (1422, Tocris), 1-EBIO (1-ethyl-1,3-dihydro-2-benzimidazolone) (SML0034, Sigma), EIPA (5-( N -Ethyl- N -isopropyl)amiloride) (A-3085, Sigma), N -methyl- d -glucamine (NMDG+ , M-2004, Sigma), ouabain (O-3125, Sigma).

    Techniques:

    Changes in 20-KDa myosin light chain (MLC 20 ) phosphorylation with the elevation of transmural pressure, and the effects of S1P or SKI application. Results are representative of immunoblots from five independent preparations. Results are expressed as means ± SEMs (n = 5). TMP: transmural pressure.

    Journal: PLoS ONE

    Article Title: The Role of Sphingosine Kinase 1/Sphingosine-1-Phosphate Pathway in the Myogenic Tone of Posterior Cerebral Arteries

    doi: 10.1371/journal.pone.0035177

    Figure Lengend Snippet: Changes in 20-KDa myosin light chain (MLC 20 ) phosphorylation with the elevation of transmural pressure, and the effects of S1P or SKI application. Results are representative of immunoblots from five independent preparations. Results are expressed as means ± SEMs (n = 5). TMP: transmural pressure.

    Article Snippet: Drugs The following drugs were used: S1P (Sigma), SKI (EIPA; Sigma, St Louis, MO, USA), NaF (Sigma), (R)-3-Amino-(3-hexylphenylamino)-4-oxobutylphosphonic acid (W146) (Sigma), 1-[1,3-Dimethyl-4-(2-methylethyl)-1H -pyrazolo[3,4-b ]pyridine-6-yl]-4-(3,5-dichloro-4-pyridinyl)-semicarbazide (JTE013) (Tocris Bioscience, Ellisville, MO, USA), 2-undecyl-thiazolidine-4-carboxylic acid (CAY10444) (Cayman Chemical, Ann Arbor, MI, USA), fasudil hydrochloride (Tocris), DPI (Sigma), apocynin (Sigma), 1-[β-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride (SKF-96365) (Tocris Bioscience), 9-phenanthrol (Sigma), and nifedipine (Sigma).

    Techniques: Western Blot

    Changes in the Ca 2+ fluorescence ratio and lumen diameter during transmural pressure elevation and S1P application. A: Representative traces demonstrate changes in the Ca 2+ ratio and lumen diameter during the elevation of transmural pressure (from 40 to 80 mmHg) and application of S1P in the absence (A 1 ) or presence (A 2 ) of SKI. B: Summarized data for changes in the Ca 2+ ratio and myogenic tone during the elevation of transmural pressure (B 1 ) and application of S1P (B 2 ) in the absence or presence of SKI. Data are expressed as means ± SEMs (n = 6). TMP: transmural pressure.

    Journal: PLoS ONE

    Article Title: The Role of Sphingosine Kinase 1/Sphingosine-1-Phosphate Pathway in the Myogenic Tone of Posterior Cerebral Arteries

    doi: 10.1371/journal.pone.0035177

    Figure Lengend Snippet: Changes in the Ca 2+ fluorescence ratio and lumen diameter during transmural pressure elevation and S1P application. A: Representative traces demonstrate changes in the Ca 2+ ratio and lumen diameter during the elevation of transmural pressure (from 40 to 80 mmHg) and application of S1P in the absence (A 1 ) or presence (A 2 ) of SKI. B: Summarized data for changes in the Ca 2+ ratio and myogenic tone during the elevation of transmural pressure (B 1 ) and application of S1P (B 2 ) in the absence or presence of SKI. Data are expressed as means ± SEMs (n = 6). TMP: transmural pressure.

    Article Snippet: Drugs The following drugs were used: S1P (Sigma), SKI (EIPA; Sigma, St Louis, MO, USA), NaF (Sigma), (R)-3-Amino-(3-hexylphenylamino)-4-oxobutylphosphonic acid (W146) (Sigma), 1-[1,3-Dimethyl-4-(2-methylethyl)-1H -pyrazolo[3,4-b ]pyridine-6-yl]-4-(3,5-dichloro-4-pyridinyl)-semicarbazide (JTE013) (Tocris Bioscience, Ellisville, MO, USA), 2-undecyl-thiazolidine-4-carboxylic acid (CAY10444) (Cayman Chemical, Ann Arbor, MI, USA), fasudil hydrochloride (Tocris), DPI (Sigma), apocynin (Sigma), 1-[β-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride (SKF-96365) (Tocris Bioscience), 9-phenanthrol (Sigma), and nifedipine (Sigma).

    Techniques: Fluorescence

    The effect of endogenous and exogenous S1P on myogenic tone. A: Representative traces showing the effect of 5 µmol/L SKI on pressure-induced myogenic tone. B: The mean data for the effects of SKI (B 1 ; n = 9), NaF (B 2 ; n = 6), and S1P (B 3 ; n = 5) on pressure-induced myogenic tone. Changes in the lumen diameter were measured in response to 20 mmHg stepwise increases in transmural pressure in Ca 2+ -containing Kreb’s-Henseleit solution (active tone; KH) or Ca 2+ -free KH solution (passive tone). C: Representative traces from five independent results showing the effect of pre-treatment of S1P on SKI-induced inhibitory effect of myogenic tone. D: Summarized data for changes in myogenic tone during the elevation of transmural pressure (from 40 mmHg to 80 mmHg) and application of S1P in posterior cerebral arteries with and without endothelium. S1P and inhibitors were administered at a transmural pressure of 40 mmHg 30 min before increasing in luminal pressure. Data are expressed as means ± SEMs (n = 5–9) and are normalized to myogenic tone at a pressure of 40 mmHg. *Significantly different compared to the control ( P

    Journal: PLoS ONE

    Article Title: The Role of Sphingosine Kinase 1/Sphingosine-1-Phosphate Pathway in the Myogenic Tone of Posterior Cerebral Arteries

    doi: 10.1371/journal.pone.0035177

    Figure Lengend Snippet: The effect of endogenous and exogenous S1P on myogenic tone. A: Representative traces showing the effect of 5 µmol/L SKI on pressure-induced myogenic tone. B: The mean data for the effects of SKI (B 1 ; n = 9), NaF (B 2 ; n = 6), and S1P (B 3 ; n = 5) on pressure-induced myogenic tone. Changes in the lumen diameter were measured in response to 20 mmHg stepwise increases in transmural pressure in Ca 2+ -containing Kreb’s-Henseleit solution (active tone; KH) or Ca 2+ -free KH solution (passive tone). C: Representative traces from five independent results showing the effect of pre-treatment of S1P on SKI-induced inhibitory effect of myogenic tone. D: Summarized data for changes in myogenic tone during the elevation of transmural pressure (from 40 mmHg to 80 mmHg) and application of S1P in posterior cerebral arteries with and without endothelium. S1P and inhibitors were administered at a transmural pressure of 40 mmHg 30 min before increasing in luminal pressure. Data are expressed as means ± SEMs (n = 5–9) and are normalized to myogenic tone at a pressure of 40 mmHg. *Significantly different compared to the control ( P

    Article Snippet: Drugs The following drugs were used: S1P (Sigma), SKI (EIPA; Sigma, St Louis, MO, USA), NaF (Sigma), (R)-3-Amino-(3-hexylphenylamino)-4-oxobutylphosphonic acid (W146) (Sigma), 1-[1,3-Dimethyl-4-(2-methylethyl)-1H -pyrazolo[3,4-b ]pyridine-6-yl]-4-(3,5-dichloro-4-pyridinyl)-semicarbazide (JTE013) (Tocris Bioscience, Ellisville, MO, USA), 2-undecyl-thiazolidine-4-carboxylic acid (CAY10444) (Cayman Chemical, Ann Arbor, MI, USA), fasudil hydrochloride (Tocris), DPI (Sigma), apocynin (Sigma), 1-[β-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride (SKF-96365) (Tocris Bioscience), 9-phenanthrol (Sigma), and nifedipine (Sigma).

    Techniques:

    Macropinocytotic internalization of Ebola virions is GP-dependent. (A) Co-localization of SNX5 with VSV pseudotyped with EBOV GP. Labeled VSV particles pseudotyped with EBOV GP (DiI-VSVΔ*G-GP) or VSV G (DiI-VSVΔ*G-G) were adsorbed to eGFP-SNX5-expressing Vero cells for 30 min on ice. The cells were then incubated at 37°C and time-lapse images were acquired at 20-second intervals over a period of 30 min by using confocal laser scanning microscope. Still frames of DiI-VSVΔ*G-GP (left panel) and DiI-VSVΔ*G-G (right panel) at 10 min after the temperature shift are shown. DiI-pseudovirions that co-localize with eGFP-SNX5 are indicated by arrows. Scale bars, 10 µm. (B) Graphic representation of the co-localization of EBOV GP-pseudotyped VSV virions with Rab7-positive vesicles. Co-localization of DiI-VSVΔ*G-GP (green bars) with Rab7-positive vesicles was analyzed at the indicated time points as indicated in the Materials and Methods . Experiments were performed in triplicate and the results are presented as the mean ± standard deviation. Results obtained for DiI-EbolaΔVP30 (blue bars) and DiI-VSVΔ*G-G (red bars) are shown for comparison. (C) Effect of macropinocytosis inhibitors on the co-localization of DiI-labeled VSV pseudovirions with eGFP-Rab7-positive vesicles. Vero cells expressing eGFP-Rab7 were pretreated with CytoD, Wort, LY294002 or EIPA for 30 min at 37°C; control cells were treated with DMSO. DiI-labeled VSVΔ*G-GP (green bars) or VSVΔ*G-G (red bars) were adsorbed to cells for 30 min on ice. The cells were then incubated at 37°C in the presence of inhibitors for 2 h. Co-localization of DiI-pseudovirions with eGFP-Rab7-positive vesicles was analyzed as described in the Materials and Methods . Experiments were carried out in triplicate and the results are presented as the mean ± standard deviation. (D) Effect of macropinocytosis inhibitors on the infectivity of VSV pseudovirions. Vero cells were treated with individual inhibitors for 30 min at 37°C and infected with VSVΔG*-GP (green bars) or VSVΔG*-G (red bars) in the presence of the inhibitor. 1 h post-infection, surface-bound virions were removed by trypsin and the cells were cultured for 24 h in the absence of inhibitors. The infection efficiency of each pseudovirus was determined by measuring the number of GFP-positive cells using with conventional fluorescent microscope. Each experiment was performed in triplicate and the relative infection efficiencies are presented as the mean ± SD.

    Journal: PLoS Pathogens

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

    doi: 10.1371/journal.ppat.1001121

    Figure Lengend Snippet: Macropinocytotic internalization of Ebola virions is GP-dependent. (A) Co-localization of SNX5 with VSV pseudotyped with EBOV GP. Labeled VSV particles pseudotyped with EBOV GP (DiI-VSVΔ*G-GP) or VSV G (DiI-VSVΔ*G-G) were adsorbed to eGFP-SNX5-expressing Vero cells for 30 min on ice. The cells were then incubated at 37°C and time-lapse images were acquired at 20-second intervals over a period of 30 min by using confocal laser scanning microscope. Still frames of DiI-VSVΔ*G-GP (left panel) and DiI-VSVΔ*G-G (right panel) at 10 min after the temperature shift are shown. DiI-pseudovirions that co-localize with eGFP-SNX5 are indicated by arrows. Scale bars, 10 µm. (B) Graphic representation of the co-localization of EBOV GP-pseudotyped VSV virions with Rab7-positive vesicles. Co-localization of DiI-VSVΔ*G-GP (green bars) with Rab7-positive vesicles was analyzed at the indicated time points as indicated in the Materials and Methods . Experiments were performed in triplicate and the results are presented as the mean ± standard deviation. Results obtained for DiI-EbolaΔVP30 (blue bars) and DiI-VSVΔ*G-G (red bars) are shown for comparison. (C) Effect of macropinocytosis inhibitors on the co-localization of DiI-labeled VSV pseudovirions with eGFP-Rab7-positive vesicles. Vero cells expressing eGFP-Rab7 were pretreated with CytoD, Wort, LY294002 or EIPA for 30 min at 37°C; control cells were treated with DMSO. DiI-labeled VSVΔ*G-GP (green bars) or VSVΔ*G-G (red bars) were adsorbed to cells for 30 min on ice. The cells were then incubated at 37°C in the presence of inhibitors for 2 h. Co-localization of DiI-pseudovirions with eGFP-Rab7-positive vesicles was analyzed as described in the Materials and Methods . Experiments were carried out in triplicate and the results are presented as the mean ± standard deviation. (D) Effect of macropinocytosis inhibitors on the infectivity of VSV pseudovirions. Vero cells were treated with individual inhibitors for 30 min at 37°C and infected with VSVΔG*-GP (green bars) or VSVΔG*-G (red bars) in the presence of the inhibitor. 1 h post-infection, surface-bound virions were removed by trypsin and the cells were cultured for 24 h in the absence of inhibitors. The infection efficiency of each pseudovirus was determined by measuring the number of GFP-positive cells using with conventional fluorescent microscope. Each experiment was performed in triplicate and the relative infection efficiencies are presented as the mean ± SD.

    Article Snippet: Dynasore, Cytochalasin D, Wortmannin, LY-294002 hydrochloride, EIPA, and Staurosporine were purchased from Sigma-Aldrich (St. Louis, USA).

    Techniques: Labeling, Expressing, Incubation, Laser-Scanning Microscopy, Standard Deviation, Infection, Cell Culture, Microscopy

    Effect of macropinocytosis inhibitors on the co-localization of DiI-labeled viral particles with Rab7-positive vesicles. Vero cells expressing eGFP-Rab7 were pretreated with cytochalasin D (CytoD), wortmannin (Wort), LY294002, or EIPA for 30 min at 37°C as described in the Materials and Methods . DiI-EbolaΔVP30 virions, DiI-Ebola VLPs and DiI-influenza virus were adsorbed to the cells for 30 min on ice. The cells were then incubated at 37°C in the presence of inhibitors for 2 h. As a control, DMSO-treated cells were incubated with labeled EBOV particles. Representative images of the co-localization of DiI-EbolaΔVP30 virions with eGFP-Rab7 acquired 2 h after the temperature shift are shown (A). DiI-labeled EbolaΔVP30 virions that co-localize with eGFP-Rab7-positive vesicles are indicated by arrows. Scale bars, 10 µm. (B) shows a graphic representation of the data. The number of DiI-labeled EbolaΔVP30 virions (blue bars), Ebola VLPs (yellow bars) and influenza virions (red bars) co-localized with eGFP-Rab7-positive vesicles was measured in 10 individual cells and the percentage of co-localization in the total DiI-virions is shown for each time point. Each experiment was carried out in triplicate and the results are presented as the mean ± SD.

    Journal: PLoS Pathogens

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

    doi: 10.1371/journal.ppat.1001121

    Figure Lengend Snippet: Effect of macropinocytosis inhibitors on the co-localization of DiI-labeled viral particles with Rab7-positive vesicles. Vero cells expressing eGFP-Rab7 were pretreated with cytochalasin D (CytoD), wortmannin (Wort), LY294002, or EIPA for 30 min at 37°C as described in the Materials and Methods . DiI-EbolaΔVP30 virions, DiI-Ebola VLPs and DiI-influenza virus were adsorbed to the cells for 30 min on ice. The cells were then incubated at 37°C in the presence of inhibitors for 2 h. As a control, DMSO-treated cells were incubated with labeled EBOV particles. Representative images of the co-localization of DiI-EbolaΔVP30 virions with eGFP-Rab7 acquired 2 h after the temperature shift are shown (A). DiI-labeled EbolaΔVP30 virions that co-localize with eGFP-Rab7-positive vesicles are indicated by arrows. Scale bars, 10 µm. (B) shows a graphic representation of the data. The number of DiI-labeled EbolaΔVP30 virions (blue bars), Ebola VLPs (yellow bars) and influenza virions (red bars) co-localized with eGFP-Rab7-positive vesicles was measured in 10 individual cells and the percentage of co-localization in the total DiI-virions is shown for each time point. Each experiment was carried out in triplicate and the results are presented as the mean ± SD.

    Article Snippet: Dynasore, Cytochalasin D, Wortmannin, LY-294002 hydrochloride, EIPA, and Staurosporine were purchased from Sigma-Aldrich (St. Louis, USA).

    Techniques: Labeling, Expressing, Incubation

    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

    Actomyosin network dynamics are required for DYNA-IND entry by IAV. (A and B) Compounds affecting actin polymerization/de-polymerization (20 µM cytochalasin D [CYTD], 20 µM cytochalasin B [CYTB], 1 µM Latrunculin A [LATR], 1 µM Jasplakinolide [JASP]), inhibitors of MLCK (5 µM ML-7 and 5 µM ML-9) and an inhibitor of myosin II, (80 µM Blebbistatin [BLEB]) were examined for their effect on DYNA-IND IAV entry in PBS (A) or DYNA-DEP IAV entry (in PBS, 10% FCS and 80 µM dynasor) (B) using the Gluc-entry assay (HeLa cells; strain WSN; MOI 0.5; incubation with inhibitors from 1 hr prior to infection to 2 hrs p.i.). Luciferase activity was determined 16 hrs p.i. (RLU plotted on the y-axis relative to the activity obtained in absence of compounds affecting the actomysoin network (grey bars, 100%). (C) An example of confocal images of HeLa cells, expressing a wildtype Rab5-GFP fusion protein (Rab5 wt) (upper panel, green fluorescence identifies transfected cells) or a dominant-negative mutant of a Rab5-GFP fusion protein (RAB5 DN; lower panel), inoculated with IAV (strain WSN; MOI 1). Infection was performed for 4 hrs after which cells were fixed and stained (red staining with a monoclonal antibody directed against NP identifies infected cells; In the example of panel C infection was performed in PBS in order to examine the DYNA-DEP entry pathway) Similar experiments were performed for GFP fusion proteins with dominant-negative and wildtype MLCK (MLCK DN and MLCK wt) and Myosin II-tail domain (MyoII-tail) or MyosinII-head domain (MyoII-head). Transfected cells were infected under DYNA-DEP (PBS) and DYNA-IND (PBS, 10% FCS and 80 µM dynasor) entry conditions. (D–F) Results were quantified by counting > 100 cells (experiment performed in triplo; transfected cells, infected cells and cells that were transfected as well as infected were counted). Relative infection of transfected cells was plotted (taking infection of non-transfected cells in the same sample as 100%). Thus, whereas Rab5 wt transfected cells are not significantly reduced or enhanced in infection compared to non-transfected cells (D, black bars), Rab5 DN transfected cells (D, grey bars) are infected at lower levels both under DYNA-DEP and DYNA-IND entry conditions. In contrast, cells transfected with MCLK DN (E) or MyoII-head (F) were infected at significantly lower levels via the DYNA-IND pathway while the DYNA-DEP pathway was not affected. Transfection efficiency for the different constructs was as follows: RAB5 wt (61%); RAB5 DN (45%); MLCK wt (50%); MLCK DN (49%); MyoII-tail (69%); MyoII-head (55%).

    Journal: PLoS Pathogens

    Article Title: Dissection of the Influenza A Virus Endocytic Routes Reveals Macropinocytosis as an Alternative Entry Pathway

    doi: 10.1371/journal.ppat.1001329

    Figure Lengend Snippet: Actomyosin network dynamics are required for DYNA-IND entry by IAV. (A and B) Compounds affecting actin polymerization/de-polymerization (20 µM cytochalasin D [CYTD], 20 µM cytochalasin B [CYTB], 1 µM Latrunculin A [LATR], 1 µM Jasplakinolide [JASP]), inhibitors of MLCK (5 µM ML-7 and 5 µM ML-9) and an inhibitor of myosin II, (80 µM Blebbistatin [BLEB]) were examined for their effect on DYNA-IND IAV entry in PBS (A) or DYNA-DEP IAV entry (in PBS, 10% FCS and 80 µM dynasor) (B) using the Gluc-entry assay (HeLa cells; strain WSN; MOI 0.5; incubation with inhibitors from 1 hr prior to infection to 2 hrs p.i.). Luciferase activity was determined 16 hrs p.i. (RLU plotted on the y-axis relative to the activity obtained in absence of compounds affecting the actomysoin network (grey bars, 100%). (C) An example of confocal images of HeLa cells, expressing a wildtype Rab5-GFP fusion protein (Rab5 wt) (upper panel, green fluorescence identifies transfected cells) or a dominant-negative mutant of a Rab5-GFP fusion protein (RAB5 DN; lower panel), inoculated with IAV (strain WSN; MOI 1). Infection was performed for 4 hrs after which cells were fixed and stained (red staining with a monoclonal antibody directed against NP identifies infected cells; In the example of panel C infection was performed in PBS in order to examine the DYNA-DEP entry pathway) Similar experiments were performed for GFP fusion proteins with dominant-negative and wildtype MLCK (MLCK DN and MLCK wt) and Myosin II-tail domain (MyoII-tail) or MyosinII-head domain (MyoII-head). Transfected cells were infected under DYNA-DEP (PBS) and DYNA-IND (PBS, 10% FCS and 80 µM dynasor) entry conditions. (D–F) Results were quantified by counting > 100 cells (experiment performed in triplo; transfected cells, infected cells and cells that were transfected as well as infected were counted). Relative infection of transfected cells was plotted (taking infection of non-transfected cells in the same sample as 100%). Thus, whereas Rab5 wt transfected cells are not significantly reduced or enhanced in infection compared to non-transfected cells (D, black bars), Rab5 DN transfected cells (D, grey bars) are infected at lower levels both under DYNA-DEP and DYNA-IND entry conditions. In contrast, cells transfected with MCLK DN (E) or MyoII-head (F) were infected at significantly lower levels via the DYNA-IND pathway while the DYNA-DEP pathway was not affected. Transfection efficiency for the different constructs was as follows: RAB5 wt (61%); RAB5 DN (45%); MLCK wt (50%); MLCK DN (49%); MyoII-tail (69%); MyoII-head (55%).

    Article Snippet: Chemicals Stocks of bafilomycin A1 (BafA1), dynasore, cytochalasin D, cytochalasin B, Blebbistatin, 17-AA-geldanamycin, ML-7, ML-9, PP-2, 5-(N-ethyl-N-isopropyl)amiloride (EIPA), IPA-3 (all obtained from Sigma-Aldrich), Latrunculin A (Enzo), jasplakinolide, wiskostatin, NSC23766 (all obtained from Calbiochem) and pirl1 (Chembridge) were prepared in dimethylsulfoxide (DMSO).

    Techniques: Incubation, Infection, Luciferase, Activity Assay, Expressing, Fluorescence, Transfection, Dominant Negative Mutation, Staining, Construct

    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

    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

    Collagen internalization is regulated by Dynamin-2, phosphoinositide-3-kinase/AKT, and actin cytoskeleton. Human HSCs were infected with K44A mutant Dynamin-2 adenovirus for 24 h or pretreated with Dynasore ( A ), blebbistatin, ML7, or Cytochalasin D ( B ), methyl-β-cyclodextrin (MβCD), filipin, nystatin, or 0.5 M sucrose ( C ), or wortmannin or AKT inhibitor ( D ) for 30 min before DQ-collagen I (0.5 μg/ml) incubation for 3 h. Cells were subjected to FACS analysis to measure matrix protein endocytosis ( n = 4, * P

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: Endocytosis of collagen by hepatic stellate cells regulates extracellular matrix dynamics

    doi: 10.1152/ajpcell.00086.2014

    Figure Lengend Snippet: Collagen internalization is regulated by Dynamin-2, phosphoinositide-3-kinase/AKT, and actin cytoskeleton. Human HSCs were infected with K44A mutant Dynamin-2 adenovirus for 24 h or pretreated with Dynasore ( A ), blebbistatin, ML7, or Cytochalasin D ( B ), methyl-β-cyclodextrin (MβCD), filipin, nystatin, or 0.5 M sucrose ( C ), or wortmannin or AKT inhibitor ( D ) for 30 min before DQ-collagen I (0.5 μg/ml) incubation for 3 h. Cells were subjected to FACS analysis to measure matrix protein endocytosis ( n = 4, * P

    Article Snippet: Dynasore, Cytochalasin D, blebbistatin, methyl-β-cyclodextrin (MβCD), Filipin, nystatin, 5-ethylisopropyl amiloride (EIPA), PDGF-BB, and fibroblast growth factor were obtained from Sigma-Aldrich (Dorset, UK).

    Techniques: Infection, Mutagenesis, Incubation, FACS

    RSV infection induces actin rearrangement. (A). RSV (moi ∼0.5) was bound to HeLa cells at 4°C, unbound virus was removed and cells warmed to 37°C, fixed at indicated times, and stained with phalloidin-AF488 (pseudocolored white) and anti-F-AF647 (red) antibody. Images represent Z-stack projections acquired with a confocal microscope. Arrowheads show actin blebs formed at the cell surface. (B). RSV (moi ∼30) was incubated with HeLa cells for 30 or 120 min at 37°C. Samples were processed according to the kit manufacturer's protocol (Cytoskeleton Inc.). Controls included mock-treated cells and cells either treated with F actin enhancer or F actin depolymerizing agent. (left) The F and G actin fractions were resolved by SDS-PAGE and western blots probed with anti-actin antibody. (right) Quantification of actin protein bands intensities by densitometry. (C–F). HeLa cells were pretreated with solvent (MOCK), cytochalasin D (CytoD), latrunculin A (LatA), jasplakinolide (Jas), nocodazole (Noc), taxol (Tax), NCS23766, pirl1, IPA-3, wiskostatin (Wisko), CK-869, CT04, Y24632 at indicated concentrations and each inhibitor was continuously present during following steps of the experiment: (C, E). Cells where infected with RSV (moi ∼3) or SFV-ZsGreen (moi ∼0.5) for up to 6 hours before FACS analysis of GFP expressing cells. (D, F). RSV (moi ∼3) was bound to the cells at 4°C followed by 1 h of internalization at 37°C. Cells were trypsinized, fixed and stained with anti-N-AF488 antibody, and the MFI of AF-488 measured by FACS.

    Journal: PLoS Pathogens

    Article Title: Host Cell Entry of Respiratory Syncytial Virus Involves Macropinocytosis Followed by Proteolytic Activation of the F Protein

    doi: 10.1371/journal.ppat.1003309

    Figure Lengend Snippet: RSV infection induces actin rearrangement. (A). RSV (moi ∼0.5) was bound to HeLa cells at 4°C, unbound virus was removed and cells warmed to 37°C, fixed at indicated times, and stained with phalloidin-AF488 (pseudocolored white) and anti-F-AF647 (red) antibody. Images represent Z-stack projections acquired with a confocal microscope. Arrowheads show actin blebs formed at the cell surface. (B). RSV (moi ∼30) was incubated with HeLa cells for 30 or 120 min at 37°C. Samples were processed according to the kit manufacturer's protocol (Cytoskeleton Inc.). Controls included mock-treated cells and cells either treated with F actin enhancer or F actin depolymerizing agent. (left) The F and G actin fractions were resolved by SDS-PAGE and western blots probed with anti-actin antibody. (right) Quantification of actin protein bands intensities by densitometry. (C–F). HeLa cells were pretreated with solvent (MOCK), cytochalasin D (CytoD), latrunculin A (LatA), jasplakinolide (Jas), nocodazole (Noc), taxol (Tax), NCS23766, pirl1, IPA-3, wiskostatin (Wisko), CK-869, CT04, Y24632 at indicated concentrations and each inhibitor was continuously present during following steps of the experiment: (C, E). Cells where infected with RSV (moi ∼3) or SFV-ZsGreen (moi ∼0.5) for up to 6 hours before FACS analysis of GFP expressing cells. (D, F). RSV (moi ∼3) was bound to the cells at 4°C followed by 1 h of internalization at 37°C. Cells were trypsinized, fixed and stained with anti-N-AF488 antibody, and the MFI of AF-488 measured by FACS.

    Article Snippet: Inhibitors, antibodies and plasmids The inhibitors used included: PI-103 (Alexis Biochemicals), dynasore, dyngo-4a,dynol-31-2, pitstop-2 (Ascent Scientific), pirl1 (Chembridge), wiskostatin (Enzo), CAS 879127-07-8, CAS 371942-96-7, dec-RVKR-CMK, LY294002, NSC23766, staurosporine, taxol, wortmannin, Y27632, α-PDX (Calbiochem), bafilomycin A, blebbistatin, calphostin C, chlorpromazine, cytochalasin D, EIPA, genistein, IPA-3, jasplakinolide, latrunculin A, leupeptin, ML7, monensin, NH4Cl, nocodazole, and rottlerin (Sigma).

    Techniques: Infection, Staining, Microscopy, Incubation, SDS Page, Western Blot, Indirect Immunoperoxidase Assay, FACS, Expressing

    Size-normalized acoustic scattering (SNACS) measures changes in mechanical properties of cells. a, Schematic of ‘Cortical Shell – Liquid Core’ model, where E mod is the elastic modulus of a shell. Cortical thickness (t s ) was set to 2% of the cell radius (r c ) 45 . b, Node deviation (volume-normalized) vs volume V from experiments with L1210 cells (black dots) and simulations using the model for three values of cortical elastic modulus (E mod , 4, 6 and 8 MPa, color lines). Vertical offsets of iso-elasticity lines define SNACS ( Methods ). Inset, Δ f / f from simulations with different cortical elastic modulus (4, 6 and 8 MPa, V = 900 fL). c, SNACS obtained from L1210 cells treated with inhibitors of actomyosin cortex: Latrunculin B (LatB, 0.02, 0.1, 0.4 and 1 μM, n = 381, 385, 346 and 383 cells, respectively, P = 0.036, 3.8 × 10 -5 , 1.03 × 10 -8 and 4.5 × 10 -18 , respectively), Cytochalsin D (CytoD, 1 μM, n = 332 cells, P = 1.2 × 10 -8 ), and Blebbistatin (Bleb, 50 μM, n = 349 cells, P = 0.023). Statistical comparisons (two-sided Welch’s t-test) were made to DMSO control (0.1 %, n = 337 cells). d, Representative single z-layer images of F-actin (LifeAct) from live L1210 cells before and after 1 μM LatB (n = 9 fields of views) and 1 μM CytoD (n = 12 fields of views) treatment. Scale bars, 10 μm. e, SNACS of L1210 cells after crosslinking with 4% Paraformaldehyde (PFA, 1 and 10 min exposure, n=247 and 367 cells, respectively, P = 2.0 × 10 -56 and 6.7 × 10 -175 , respectively). Statistical comparisons (two-sided Welch’s t-test) were made to DMSO control (0.1 %, n = 1047 cells). f, Effect of osmotic stress on SNACS. Cells were resuspended in hypo- (200 mOsm, n = 611 cells, P = 2.9 × 10 -10 ) or hyperosmotic (350, 400 and 500 mOsm, n = 544, 571 and 574 cells, respectively, P = 8.1 × 10 -65 , 2.6 × 10 -144 and

    Journal: Nature methods

    Article Title: Non-invasive monitoring of single-cell mechanics by acoustic scattering

    doi: 10.1038/s41592-019-0326-x

    Figure Lengend Snippet: Size-normalized acoustic scattering (SNACS) measures changes in mechanical properties of cells. a, Schematic of ‘Cortical Shell – Liquid Core’ model, where E mod is the elastic modulus of a shell. Cortical thickness (t s ) was set to 2% of the cell radius (r c ) 45 . b, Node deviation (volume-normalized) vs volume V from experiments with L1210 cells (black dots) and simulations using the model for three values of cortical elastic modulus (E mod , 4, 6 and 8 MPa, color lines). Vertical offsets of iso-elasticity lines define SNACS ( Methods ). Inset, Δ f / f from simulations with different cortical elastic modulus (4, 6 and 8 MPa, V = 900 fL). c, SNACS obtained from L1210 cells treated with inhibitors of actomyosin cortex: Latrunculin B (LatB, 0.02, 0.1, 0.4 and 1 μM, n = 381, 385, 346 and 383 cells, respectively, P = 0.036, 3.8 × 10 -5 , 1.03 × 10 -8 and 4.5 × 10 -18 , respectively), Cytochalsin D (CytoD, 1 μM, n = 332 cells, P = 1.2 × 10 -8 ), and Blebbistatin (Bleb, 50 μM, n = 349 cells, P = 0.023). Statistical comparisons (two-sided Welch’s t-test) were made to DMSO control (0.1 %, n = 337 cells). d, Representative single z-layer images of F-actin (LifeAct) from live L1210 cells before and after 1 μM LatB (n = 9 fields of views) and 1 μM CytoD (n = 12 fields of views) treatment. Scale bars, 10 μm. e, SNACS of L1210 cells after crosslinking with 4% Paraformaldehyde (PFA, 1 and 10 min exposure, n=247 and 367 cells, respectively, P = 2.0 × 10 -56 and 6.7 × 10 -175 , respectively). Statistical comparisons (two-sided Welch’s t-test) were made to DMSO control (0.1 %, n = 1047 cells). f, Effect of osmotic stress on SNACS. Cells were resuspended in hypo- (200 mOsm, n = 611 cells, P = 2.9 × 10 -10 ) or hyperosmotic (350, 400 and 500 mOsm, n = 544, 571 and 574 cells, respectively, P = 8.1 × 10 -65 , 2.6 × 10 -144 and

    Article Snippet: Chemical concentrations used were 0.1% DMSO (controls, Sigma-Aldrich), 0.02 – 1 µM Latrunculin B (Sigma-Aldrich), 1 µM Cytochalsin D (Sigma-Aldrich), 25 µM (inhibiting Cytokinesis, long-term traces) or 50 µM (Actomyosin cortex disruption, end-point assay) Blebbistatin (Sigma-Aldrich), 10 µM EIPA (Sigma-Aldrich), 5 µM STLC (Sigma-Aldrich) and 2 µM RO3306 (R & D Systems).

    Techniques:

    PHEV propagation depends upon cholesterol fluidity but does not involve caveola/raft-dependent endocytosis. (A) PHEV entry was assessed in cells treated with DMSO (control), MβCD (5 mM), Nys/Prog (30/10 μM), or genistein (100 μM) for 60 min. Following binding on ice, PHEV was allowed to internalize at 37°C for 60 min. Surface-bound virus was removed, and the cells were fixed and visualized by use of a confocal microscope. (B) Quantitative results for the average PHEV and CTB fluorescence intensities in cells pretreated with pharmacological inhibitors are presented in a histogram. (C) PHEV internalization after treatment of Neuro-2a cells with the indicated agents for 1 h was quantified by qRT-PCR. Data are shown as percentages of PHEV uptake compared to that of control-treated cells. (D) PHEV infection assays were carried out with MβCD-, Nys/Prog-, and genistein-treated cells. PHEV genome equivalents were estimated by qRT-PCR, using the 2 −ΔΔ CT method, while viral protein synthesis levels were estimated by Western blotting with anti-PHEV-S antibody. GAPDH was used as a loading control. (E) A PHEV uptake and propagation assay was carried out with siCav1-transfected Neuro-2a cells. Infected cells were lysed to quantitate viral RNA copy numbers by qRT-PCR, and the silencing efficiency of siCav1 was analyzed by Western blotting using anti-caveolin-1 antibody. (F) The lack of involvement of the caveola/lipid raft-mediated route was demonstrated by the stability of PHEV infection in siCav1-transfected cells. Pretransfected cells were incubated with PHEV for 24 h to allow virus propagation. The cells were then fixed, stained with anti-Cav1 (red) and anti-PHEV (green) antibodies, and visualized by confocal microscopy. Data shown are means ± SD for three independent experiments. *, P

    Journal: Journal of Virology

    Article Title: Porcine Hemagglutinating Encephalomyelitis Virus Enters Neuro-2a Cells via Clathrin-Mediated Endocytosis in a Rab5-, Cholesterol-, and pH-Dependent Manner

    doi: 10.1128/JVI.01083-17

    Figure Lengend Snippet: PHEV propagation depends upon cholesterol fluidity but does not involve caveola/raft-dependent endocytosis. (A) PHEV entry was assessed in cells treated with DMSO (control), MβCD (5 mM), Nys/Prog (30/10 μM), or genistein (100 μM) for 60 min. Following binding on ice, PHEV was allowed to internalize at 37°C for 60 min. Surface-bound virus was removed, and the cells were fixed and visualized by use of a confocal microscope. (B) Quantitative results for the average PHEV and CTB fluorescence intensities in cells pretreated with pharmacological inhibitors are presented in a histogram. (C) PHEV internalization after treatment of Neuro-2a cells with the indicated agents for 1 h was quantified by qRT-PCR. Data are shown as percentages of PHEV uptake compared to that of control-treated cells. (D) PHEV infection assays were carried out with MβCD-, Nys/Prog-, and genistein-treated cells. PHEV genome equivalents were estimated by qRT-PCR, using the 2 −ΔΔ CT method, while viral protein synthesis levels were estimated by Western blotting with anti-PHEV-S antibody. GAPDH was used as a loading control. (E) A PHEV uptake and propagation assay was carried out with siCav1-transfected Neuro-2a cells. Infected cells were lysed to quantitate viral RNA copy numbers by qRT-PCR, and the silencing efficiency of siCav1 was analyzed by Western blotting using anti-caveolin-1 antibody. (F) The lack of involvement of the caveola/lipid raft-mediated route was demonstrated by the stability of PHEV infection in siCav1-transfected cells. Pretransfected cells were incubated with PHEV for 24 h to allow virus propagation. The cells were then fixed, stained with anti-Cav1 (red) and anti-PHEV (green) antibodies, and visualized by confocal microscopy. Data shown are means ± SD for three independent experiments. *, P

    Article Snippet: All endocytic inhibitors, i.e., chlorpromazine (CPZ), genistein, methyl-β-cyclodextrin (MβCD), nystatin (Nys), progesterone (Prog), ammonium chloride (NH4 Cl), chloroquine (CQ), cytochalasin D (CytoD), 5-(N -ethyl-N -isopropyl) amiloride (EIPA), and dynasore, were purchased from Sigma.

    Techniques: Binding Assay, Microscopy, CtB Assay, Fluorescence, Quantitative RT-PCR, Infection, Western Blot, Transfection, Incubation, Staining, Confocal Microscopy