bafilomycin a1 Millipore Search Results


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  • 95
    Millipore bafilomycin a1
    20A promotes autophagy induction through a mechanism that involves ATM activation. ( A ) HeLa cells were treated for the indicated times with 6 μM 20A . The activities of the MTORC1 and AMPK pathways were determined by immunoblot analysis using antibodies directed against phospho-4EBP1 (Thr37/46), 4EBP1, phospho-p70 (Thr 389), p70, phospho-AMPK (Thr 172) and AMPK. ( B ) Representative electron micrographs of HeLa cells untreated or treated for 16 h with 6 μM 20A . ( C ) Left, immunofluorescence analysis for the abundance of LC3 puncta (green dots) in HeLa cells after 3 h in either the absence or presence of 6 μM 20A ; Hoechst was used to stain nuclei (blue). Representative confocal images are shown; scale bar = 10 μm. Center, immunoblot images of LC3-II levels in HeLa cells treated with 6 μM 20A for the indicated times. Right, immunoblot of cells treated with or without E64d (10 μg/ml) plus pepstatin A (2 μg/ml) 2 h prior 20A treatment (6 μM, 8 h). ( D ) Immunoblot analysis of phospho-p62 (Ser 403) and p62 levels in HeLa cells treated with 6 μM 20A for the indicated times. ( E ) Immunoblot analysis of LC3-II levels in HeLa cells incubated with or without 10 μM KU55933 for 2 h and then treated with 6 μM 20A for 8 h. Where indicated, cells were incubated with 10 μg/ml E64d plus 2 μg/ml pepstatin A for 2 h prior 20A treatment. Where noted, <t>bafilomycin</t> A1 (50 nM) was added 3 h before cell lysis.
    Bafilomycin A1, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 5807 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Tocris bafilomycin a1
    Deletion of ATG7 sensitizes cells to starvation without impacting proliferation under nutrient-replete conditions. ( A . ATG7-WT and ATG7-null clones 17 and 47 were treated for 24 h in the absence or presence of 50 ng/mL <t>bafilomycin</t> A1 and steady-state levels of endogenous markers visualized by Western blot. ( B ) Cells were cultured for 5 d in six-well dishes in regular growth medium (RPMI/FBS) or were starved for 3 d in HBSS followed by recovery for 2 d in regular growth medium. Cells then were fixed and stained with Crystal Violet. ( C ) Cells were grown in 96-well plates, and confluence was measured at the indicated time points by the IncuCyte imaging system. ( D ) A549 ATG7 wild-type or ATG7-null clonal cell lines generated by TALEN genomic editing were treated with or without 10 nM bafilomycin A1 for 24 h and then were lysed and assessed for modulation of ATG7 function. Vinculin was probed as a loading control. ( E ) A549 ATG7 wild-type and ATG7-null cell lines seeded in six-well dishes were maintained in regular growth medium (DMEM/FBS) or were subjected to starvation in HBSS for 5 d followed by recovery. Cells were stained with sulforhodamine B (SRB). ( F ) Equal cell numbers of A549 ATG7 wild-type or ATG7-null cells were plated, and proliferation was assessed after 4 d by SRB staining.
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    99
    Merck KGaA bafilomycin a1
    Effect of mAbs on levels of endogenous AQP4 in primary cultured astrocytes. (A) Representative immunoblots of lysates treated with 1 µg/ml of C9401 (lane 2), E5415A (lanes 3 and 4), or E5415B (lanes 5 and 6) in the absence (lanes 2, 3, and 5) or presence (lanes 4 and 6) of 500 nM <t>bafilomycin</t> A1 at 37 °C for 24 h using anti-AQP4 (upper panel) or anti-actin (lower panel) antibody. (B) Effect of E5415A or E5415B on levels of endogenous AQP4 (black column) and actin (grey column). Values are determined by Western blotting and estimated as fold relative to non-treated cells (A, lane 1). **( P
    Bafilomycin A1, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 99/100, based on 81 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Millipore autophagy bafilomycin a1
    Effect of mAbs on levels of endogenous AQP4 in primary cultured astrocytes. (A) Representative immunoblots of lysates treated with 1 µg/ml of C9401 (lane 2), E5415A (lanes 3 and 4), or E5415B (lanes 5 and 6) in the absence (lanes 2, 3, and 5) or presence (lanes 4 and 6) of 500 nM <t>bafilomycin</t> A1 at 37 °C for 24 h using anti-AQP4 (upper panel) or anti-actin (lower panel) antibody. (B) Effect of E5415A or E5415B on levels of endogenous AQP4 (black column) and actin (grey column). Values are determined by Western blotting and estimated as fold relative to non-treated cells (A, lane 1). **( P
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    Millipore vatpase inhibitor bafilomycin a1
    Model for the relief of BST-2-mediated restriction by Vpu. (A) Vpu recruits β-TrCP to induce ubiquitin-mediated trafficking events that remove BST-2 from the plasma membrane, its site of action as a virion-tethering factor. Circles in the cytoplasmic domain of Vpu represent phosphoserines 52 and 56. The interaction between BST-2 and Vpu and the ubiquitination of BST-2 are currently speculative. (B) Vpu induces <t>bafilomycin</t> A1-sensitive post-endocytic trafficking of BST-2 and endo-lysosomal degradation. The removal of BST-2 from the plasma membrane involves constitutive endocytosis of BST-2 via AP-2, followed by Vpu-mediated post-endocytic sorting events. Recycling of BST-2 to the plasma membrane in the absence of Vpu is currently speculative.
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    Millipore lysosomotropic agent bafilomycin a1
    Model for the relief of BST-2-mediated restriction by Vpu. (A) Vpu recruits β-TrCP to induce ubiquitin-mediated trafficking events that remove BST-2 from the plasma membrane, its site of action as a virion-tethering factor. Circles in the cytoplasmic domain of Vpu represent phosphoserines 52 and 56. The interaction between BST-2 and Vpu and the ubiquitination of BST-2 are currently speculative. (B) Vpu induces <t>bafilomycin</t> A1-sensitive post-endocytic trafficking of BST-2 and endo-lysosomal degradation. The removal of BST-2 from the plasma membrane involves constitutive endocytosis of BST-2 via AP-2, followed by Vpu-mediated post-endocytic sorting events. Recycling of BST-2 to the plasma membrane in the absence of Vpu is currently speculative.
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    Millipore 200 n m bafilomycin a1
    TMR-r13 does not utilize the endocytic pathway to access the cytosolic space of cells. A, inhibitors of endocytic processes do not prevent cytosolic penetration. MCH58 cells were pretreated with 50 μ m amiloride, 200 n m <t>bafilomycin,</t> or PBS supplemented
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    Millipore lysosome inhibitor bafilomycin a1
    TMR-r13 does not utilize the endocytic pathway to access the cytosolic space of cells. A, inhibitors of endocytic processes do not prevent cytosolic penetration. MCH58 cells were pretreated with 50 μ m amiloride, 200 n m <t>bafilomycin,</t> or PBS supplemented
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    Millipore autophagy inhibitor bafilomycin a1
    ARPE-19 cells were treated with 288 ng Resvega, 50 nM <t>bafilomycin</t> A1, or their combination for 12 h ( A ) in normal growth conditions and ( B ) a starvation-induced autophagy model. The protein level of p62 and LC3-I/LC3-II were analyzed by Western blot, while the expression was quantified in a comparison to α-tubulin and presented as a fold change compared to control. Western blotting data are shown as mean ± SD ( n = 3). * p
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    Millipore lysosomal proteolysis bafilomycin a1
    ARPE-19 cells were treated with 288 ng Resvega, 50 nM <t>bafilomycin</t> A1, or their combination for 12 h ( A ) in normal growth conditions and ( B ) a starvation-induced autophagy model. The protein level of p62 and LC3-I/LC3-II were analyzed by Western blot, while the expression was quantified in a comparison to α-tubulin and presented as a fold change compared to control. Western blotting data are shown as mean ± SD ( n = 3). * p
    Lysosomal Proteolysis Bafilomycin A1, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore vatpase bafilomycin a1
    ARPE-19 cells were treated with 288 ng Resvega, 50 nM <t>bafilomycin</t> A1, or their combination for 12 h ( A ) in normal growth conditions and ( B ) a starvation-induced autophagy model. The protein level of p62 and LC3-I/LC3-II were analyzed by Western blot, while the expression was quantified in a comparison to α-tubulin and presented as a fold change compared to control. Western blotting data are shown as mean ± SD ( n = 3). * p
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    Millipore autophagy flux assessment bafilomycin a1
    ARPE-19 cells were treated with 288 ng Resvega, 50 nM <t>bafilomycin</t> A1, or their combination for 12 h ( A ) in normal growth conditions and ( B ) a starvation-induced autophagy model. The protein level of p62 and LC3-I/LC3-II were analyzed by Western blot, while the expression was quantified in a comparison to α-tubulin and presented as a fold change compared to control. Western blotting data are shown as mean ± SD ( n = 3). * p
    Autophagy Flux Assessment Bafilomycin A1, supplied by Millipore, used in various techniques. Bioz Stars score: 76/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore lysosomal atpase inhibitor bafilomycin a1
    ARPE-19 cells were treated with 288 ng Resvega, 50 nM <t>bafilomycin</t> A1, or their combination for 12 h ( A ) in normal growth conditions and ( B ) a starvation-induced autophagy model. The protein level of p62 and LC3-I/LC3-II were analyzed by Western blot, while the expression was quantified in a comparison to α-tubulin and presented as a fold change compared to control. Western blotting data are shown as mean ± SD ( n = 3). * p
    Lysosomal Atpase Inhibitor Bafilomycin A1, supplied by Millipore, used in various techniques. Bioz Stars score: 83/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore h pump inhibitor bafilomycin a1
    ARPE-19 cells were treated with 288 ng Resvega, 50 nM <t>bafilomycin</t> A1, or their combination for 12 h ( A ) in normal growth conditions and ( B ) a starvation-induced autophagy model. The protein level of p62 and LC3-I/LC3-II were analyzed by Western blot, while the expression was quantified in a comparison to α-tubulin and presented as a fold change compared to control. Western blotting data are shown as mean ± SD ( n = 3). * p
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    Millipore c127 bafilomycin a1
    ARPE-19 cells were treated with 288 ng Resvega, 50 nM <t>bafilomycin</t> A1, or their combination for 12 h ( A ) in normal growth conditions and ( B ) a starvation-induced autophagy model. The protein level of p62 and LC3-I/LC3-II were analyzed by Western blot, while the expression was quantified in a comparison to α-tubulin and presented as a fold change compared to control. Western blotting data are shown as mean ± SD ( n = 3). * p
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    Millipore autophagic flux inhibitor bafilomycin a1
    Autophagy is activated during LSC’s stress response to UVA. (A) Representative images of autophagosomes in ATG7KD LSCs under UVA stress. Arrowheads show <t>autophagic</t> cells, asterisks indicate absence of autophagosomes. Scale bar, 50 μm. (B) Quantification of cells with autophagic activity in response to UVA. (C) Representative western blot image of autophagic flux in UVA-irradiated LSC colonies in absence and presence of <t>BafA1,</t> with or without UVA. LC3B-I and II were detected by immunoblotting at indicated time points. (D) Densitometric analysis of LC3B-II expression normalized to GAPDH. n = 3, * p
    Autophagic Flux Inhibitor Bafilomycin A1, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore stz rh bafilomycin a1
    H 2 O 2 -induced oxidative stress impairs autophagic flux and decreases SIRT1 activity in H9C2 cells. H9C2 cells were treated with concentrations of H 2 O 2 for 12 hrs with or without <t>bafilomycin</t> A1 (Bafilo A1; 100 nM). Western blot analysis of protein levels of p62, cleaved caspase 3 (C-Caspase3) ( A ), SIRT1 ( C ), Ac-FOXO1, and total FOXO1 ( D ). Data are mean ± SEM. * P
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    Millipore bafilomycin a1 bafa1 inhibitor of v atpase calbiochem bad soden germany
    H 2 O 2 -induced oxidative stress impairs autophagic flux and decreases SIRT1 activity in H9C2 cells. H9C2 cells were treated with concentrations of H 2 O 2 for 12 hrs with or without <t>bafilomycin</t> A1 (Bafilo A1; 100 nM). Western blot analysis of protein levels of p62, cleaved caspase 3 (C-Caspase3) ( A ), SIRT1 ( C ), Ac-FOXO1, and total FOXO1 ( D ). Data are mean ± SEM. * P
    Bafilomycin A1 Bafa1 Inhibitor Of V Atpase Calbiochem Bad Soden Germany, supplied by Millipore, used in various techniques. Bioz Stars score: 84/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    Millipore virus entry replication bafilomycin a1
    HCV transmission by exosomes and free virus can be blocked by proton pump inhibitor (Lansoprazole) and Vacuolar-type H+-ATPase inhibitor <t>(bafilomycin</t> A1). (A B) Huh 7.5 cells were pre-treated with Lansoprazole (2.5 µg/ml, 5 µg/ml, and 10 µg/ml) for 1 h at indicated concentrations, and then infected with (A) HCV exosomes or (B) cell free HCV virus for 24 h. Total protein was then extracted from cells and analyzed for HCV NS3 protein. (C D) Huh 7.5 cells were pre-treated with bafilomycin A1(Baf A1) (12.5 nM, 25 nM, 50 nM, and 100 nM) for one hour at concentrations indicated, then infected with (C) HCV J6/JFH-1 exosomes or (D) cell free HCV virus for 24 h. Total RNA was then extracted from cells and analyzed for HCV RNA. An MOI of 1 of HCV-exosomes and cell free HCV virus were used for all infections. Data presented here is representative of 3 independent experiments with p
    Virus Entry Replication Bafilomycin A1, supplied by Millipore, used in various techniques. Bioz Stars score: 77/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore vacuolar atpase inhibitor bafilomycin a1
    HCV transmission by exosomes and free virus can be blocked by proton pump inhibitor (Lansoprazole) and Vacuolar-type H+-ATPase inhibitor <t>(bafilomycin</t> A1). (A B) Huh 7.5 cells were pre-treated with Lansoprazole (2.5 µg/ml, 5 µg/ml, and 10 µg/ml) for 1 h at indicated concentrations, and then infected with (A) HCV exosomes or (B) cell free HCV virus for 24 h. Total protein was then extracted from cells and analyzed for HCV NS3 protein. (C D) Huh 7.5 cells were pre-treated with bafilomycin A1(Baf A1) (12.5 nM, 25 nM, 50 nM, and 100 nM) for one hour at concentrations indicated, then infected with (C) HCV J6/JFH-1 exosomes or (D) cell free HCV virus for 24 h. Total RNA was then extracted from cells and analyzed for HCV RNA. An MOI of 1 of HCV-exosomes and cell free HCV virus were used for all infections. Data presented here is representative of 3 independent experiments with p
    Vacuolar Atpase Inhibitor Bafilomycin A1, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore v atpase inhibitor bafilomycin a1
    Ece1-independent phagosome maturation. a IL-1β levels measured by ELISA in culture supernatants of LPS-primed hMDMs. Cells were treated with synthetic Candidalysin for 5 h. Selected samples were pre-treated with the vacuolar H+ <t>ATPase</t> inhibitor <t>Bafilomycin</t> A1 1 h prior to administration of synthetic Candidalysin. b , c Human MDMs were infected with C . albicans Wt, re-integrant ( ece1 Δ/Δ + ECE1 ) or mutant strains ( ece1 Δ/Δ, ece1 Δ/Δ + ECE1 Δ 184–279 ) (MOI 5) and co-localization of C . albicans -containing phagosomes with b the phagosomal marker LAMP1 or c the lysosomal acidification marker LysoTracker was quantified at indicated time points. d Human MDMs pre-stained with LysoTracker were infected with heat-killed C . albicans Wt (MOI 5) for 3 h in presence or absence of Bafilomycin A1 (phagosomal acidification inhibitor) or synthetic Candidalysin. C . albicans -containing phagosomes were quantified for the percentage of LysoTracker-positive phagosomes and LysoTracker intensity. e Murine RAW264.7 Dectin-1 macrophages were infected with C . albicans Wt or ece1 Δ/Δ mutant yeasts (MOI 2) and co-localization of C . albicans -containing phagosomes with the phago(lyso)somal markers Lamp1, PI(4)P, and Rab7 was quantified at indicated time points. f , g Murine RAW264.7 Dectin-1 macrophages were infected with C . albicans Wt or ece1 Δ/Δ mutant strain as described in e . Representative image of Lamp1 ( f ) or PI(4)P and Rab7 ( g ) acquisition 30 min after phagocytosis. ConA staining of non-phagocytosed C . albicans . Scale bar 8 μm. Values are represented as scatterplot with median of three independent donors or experiments ( n ≥ 3)
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    Millipore bafilomycina1 bafa1 treatment
    Ece1-independent phagosome maturation. a IL-1β levels measured by ELISA in culture supernatants of LPS-primed hMDMs. Cells were treated with synthetic Candidalysin for 5 h. Selected samples were pre-treated with the vacuolar H+ <t>ATPase</t> inhibitor <t>Bafilomycin</t> A1 1 h prior to administration of synthetic Candidalysin. b , c Human MDMs were infected with C . albicans Wt, re-integrant ( ece1 Δ/Δ + ECE1 ) or mutant strains ( ece1 Δ/Δ, ece1 Δ/Δ + ECE1 Δ 184–279 ) (MOI 5) and co-localization of C . albicans -containing phagosomes with b the phagosomal marker LAMP1 or c the lysosomal acidification marker LysoTracker was quantified at indicated time points. d Human MDMs pre-stained with LysoTracker were infected with heat-killed C . albicans Wt (MOI 5) for 3 h in presence or absence of Bafilomycin A1 (phagosomal acidification inhibitor) or synthetic Candidalysin. C . albicans -containing phagosomes were quantified for the percentage of LysoTracker-positive phagosomes and LysoTracker intensity. e Murine RAW264.7 Dectin-1 macrophages were infected with C . albicans Wt or ece1 Δ/Δ mutant yeasts (MOI 2) and co-localization of C . albicans -containing phagosomes with the phago(lyso)somal markers Lamp1, PI(4)P, and Rab7 was quantified at indicated time points. f , g Murine RAW264.7 Dectin-1 macrophages were infected with C . albicans Wt or ece1 Δ/Δ mutant strain as described in e . Representative image of Lamp1 ( f ) or PI(4)P and Rab7 ( g ) acquisition 30 min after phagocytosis. ConA staining of non-phagocytosed C . albicans . Scale bar 8 μm. Values are represented as scatterplot with median of three independent donors or experiments ( n ≥ 3)
    Bafilomycina1 Bafa1 Treatment, supplied by Millipore, used in various techniques. Bioz Stars score: 81/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore late stage autophagy inhibitor bafilomycin a1
    Ece1-independent phagosome maturation. a IL-1β levels measured by ELISA in culture supernatants of LPS-primed hMDMs. Cells were treated with synthetic Candidalysin for 5 h. Selected samples were pre-treated with the vacuolar H+ <t>ATPase</t> inhibitor <t>Bafilomycin</t> A1 1 h prior to administration of synthetic Candidalysin. b , c Human MDMs were infected with C . albicans Wt, re-integrant ( ece1 Δ/Δ + ECE1 ) or mutant strains ( ece1 Δ/Δ, ece1 Δ/Δ + ECE1 Δ 184–279 ) (MOI 5) and co-localization of C . albicans -containing phagosomes with b the phagosomal marker LAMP1 or c the lysosomal acidification marker LysoTracker was quantified at indicated time points. d Human MDMs pre-stained with LysoTracker were infected with heat-killed C . albicans Wt (MOI 5) for 3 h in presence or absence of Bafilomycin A1 (phagosomal acidification inhibitor) or synthetic Candidalysin. C . albicans -containing phagosomes were quantified for the percentage of LysoTracker-positive phagosomes and LysoTracker intensity. e Murine RAW264.7 Dectin-1 macrophages were infected with C . albicans Wt or ece1 Δ/Δ mutant yeasts (MOI 2) and co-localization of C . albicans -containing phagosomes with the phago(lyso)somal markers Lamp1, PI(4)P, and Rab7 was quantified at indicated time points. f , g Murine RAW264.7 Dectin-1 macrophages were infected with C . albicans Wt or ece1 Δ/Δ mutant strain as described in e . Representative image of Lamp1 ( f ) or PI(4)P and Rab7 ( g ) acquisition 30 min after phagocytosis. ConA staining of non-phagocytosed C . albicans . Scale bar 8 μm. Values are represented as scatterplot with median of three independent donors or experiments ( n ≥ 3)
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    Effects of collagen α-5(IV) chain (Col4a5) knockdown on the <t>V-ATPase</t> activity and subunit expression in Raw264.7 macrophages. (A) Relative microsomal V-ATPase activity in control (VC) and Col4a5-knockdown (C51, C52 and C53) macrophage cell lines. (B) Colony forming units (CFUs) of mycobacterium bovis Calmette-Guérin ( BCG ) were counted in VC, C51, C52 and C53 cell lines infected with BCG at MOI 3, which were treated with the V-ATPase-specific inhibitor <t>bafilomycin</t> A1 (BA1) or in controls (Con). (C) Western blot analysis detected protein bands of the V-ATPase subunits B (VAB) and E (VAE) in VC, C51, C52 and C53 cell lines without BCG infection (MOI 0) or with BCG infection (MOI 2, 8). Tubulin served as loading control. * P
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    Effects of collagen α-5(IV) chain (Col4a5) knockdown on the <t>V-ATPase</t> activity and subunit expression in Raw264.7 macrophages. (A) Relative microsomal V-ATPase activity in control (VC) and Col4a5-knockdown (C51, C52 and C53) macrophage cell lines. (B) Colony forming units (CFUs) of mycobacterium bovis Calmette-Guérin ( BCG ) were counted in VC, C51, C52 and C53 cell lines infected with BCG at MOI 3, which were treated with the V-ATPase-specific inhibitor <t>bafilomycin</t> A1 (BA1) or in controls (Con). (C) Western blot analysis detected protein bands of the V-ATPase subunits B (VAB) and E (VAE) in VC, C51, C52 and C53 cell lines without BCG infection (MOI 0) or with BCG infection (MOI 2, 8). Tubulin served as loading control. * P
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    The stimulation-induced alkalinizing phase is selectively blocked by inhibiting exocytosis (A) and by inhibiting vesicular H + -ATPase (vATPase, B). A, Responses before and after exposure to 20 nM BoNT A (left) or 14.5 nM BoNT E (right) for 80-140 min. B, Responses before and after exposure to 1 μM folimycin (left) or 1 μM bafilomycin (right) for 1-4 hr. Folimycin produced no significant change in resting fluorescence. Ca, averaged effects of these drugs and of vesamicol on the magnitudes of the early acidifying response (measured 3-4 s following stimulation onset) and the late alkalinizing response (measured 3-4 s following cessation of stimulation). Vesamicol (7 μM) exposure was 3 hr (n=4 terminals). Results with both BoNTs were averaged together, as were results with <t>foli-</t> and <t>bafilo-mycin</t> (fol/baf). * indicates significant difference from control (p
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    Effects of <t>Baf</t> A1, a pharmacological inhibitor of virus uncoating in endosomes, on cytokine and chemokine production by primary microglia cultures prepared from wild-type mice and then exposed to NSV Some wells were treated with <t>Baf</t> A1 as outlined in Materials and Methods section. A minimum of three wells either without or with Baf A1 pretreatment were then exposed to NSV, and cytokine and chemokine concentrations measured by ELISA in culture supernatants 24 h later using ELISA-specific for IL-12p40 ( A ), IFN-α ( B ), CCL2 ( C ) and CCL5 ( D ). The production of these mediators without or with Baf A1 treatment was measured using primary cells prepared on two separate occasions.
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    CD14 + monocytes inhibit IFN-α production triggered by Ab-RSV via TLR7 in pDC. ( A ) IFN-α production in CD14 + cell depleted PBMCs induced by Ab-RSV complexes, TLR9 ligand ODN 2216 and TLR7 ligand imiquimod is decreased by blocking <t>endosomal</t> acidification with 50nM Bafilomycin A 1 . ( B ) Ab-RSV-induced IFN-α production in CD14 + cell depleted PBMCs was abrogated in the presence of immune regulatory sequence (IRS) 661 (1.4 µM) a specific blocking agent for endosomal TLR7 and not by a scrambled control nucleotide. ( C ) pDC are the source of Ab-RSV induced, TLR7 mediated production of IFN-α, as shown by intracellular staining for IFN-α in Lineage (CD3 neg. , CD14 neg. , CD19 neg. , CD16 neg. , CD56 neg. , Lin-1), MHC-II high , BDCA-4 + cells. The inhibitor brefeldin A was added at different time points post infection (the time points when BFA was added are given on the X-axis). Cytokines were allowed to accumulate for 10 hrs. after addition of BFA. ( D ) Purified pDC (obtained by negative selection removing CD3 + , CD19 + and CD16 + cells from fresh PBMC, followed by FACS purification of the BDCA-4 + cell population, which resulted in > 95% pure pDC) produce IFN-α upon infection with RSV. This response is abrogated after UV inactivation of RSV. In AS, both live RSV and UV-inactivated RSV induced IFN-α production to a similar extent. One representative experiment out of two performed with pDC isolated from two different donors is shown. ( E ) IFN-α production by Ab-RSV in purified pDC is blocked by IRS661 (1.4µM). ( F ) TLR1,-2 (PAM3CSK4, Peptidoglycan) and TLR4 (LPS) ligands suppress TLR9-triggered (ODN 2216) IFN-α production, but do not affect TLR7 (Gardiquimod) induced IFN-α production. All data represent mean ± SEM of triplicate measurements within 1 donor and analyzed using one way ANOVA followed by a Bonferroni post-test. ns not significant, *P
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    CD14 + monocytes inhibit IFN-α production triggered by Ab-RSV via TLR7 in pDC. ( A ) IFN-α production in CD14 + cell depleted PBMCs induced by Ab-RSV complexes, TLR9 ligand ODN 2216 and TLR7 ligand imiquimod is decreased by blocking <t>endosomal</t> acidification with 50nM Bafilomycin A 1 . ( B ) Ab-RSV-induced IFN-α production in CD14 + cell depleted PBMCs was abrogated in the presence of immune regulatory sequence (IRS) 661 (1.4 µM) a specific blocking agent for endosomal TLR7 and not by a scrambled control nucleotide. ( C ) pDC are the source of Ab-RSV induced, TLR7 mediated production of IFN-α, as shown by intracellular staining for IFN-α in Lineage (CD3 neg. , CD14 neg. , CD19 neg. , CD16 neg. , CD56 neg. , Lin-1), MHC-II high , BDCA-4 + cells. The inhibitor brefeldin A was added at different time points post infection (the time points when BFA was added are given on the X-axis). Cytokines were allowed to accumulate for 10 hrs. after addition of BFA. ( D ) Purified pDC (obtained by negative selection removing CD3 + , CD19 + and CD16 + cells from fresh PBMC, followed by FACS purification of the BDCA-4 + cell population, which resulted in > 95% pure pDC) produce IFN-α upon infection with RSV. This response is abrogated after UV inactivation of RSV. In AS, both live RSV and UV-inactivated RSV induced IFN-α production to a similar extent. One representative experiment out of two performed with pDC isolated from two different donors is shown. ( E ) IFN-α production by Ab-RSV in purified pDC is blocked by IRS661 (1.4µM). ( F ) TLR1,-2 (PAM3CSK4, Peptidoglycan) and TLR4 (LPS) ligands suppress TLR9-triggered (ODN 2216) IFN-α production, but do not affect TLR7 (Gardiquimod) induced IFN-α production. All data represent mean ± SEM of triplicate measurements within 1 donor and analyzed using one way ANOVA followed by a Bonferroni post-test. ns not significant, *P
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    CD14 + monocytes inhibit IFN-α production triggered by Ab-RSV via TLR7 in pDC. ( A ) IFN-α production in CD14 + cell depleted PBMCs induced by Ab-RSV complexes, TLR9 ligand ODN 2216 and TLR7 ligand imiquimod is decreased by blocking <t>endosomal</t> acidification with 50nM Bafilomycin A 1 . ( B ) Ab-RSV-induced IFN-α production in CD14 + cell depleted PBMCs was abrogated in the presence of immune regulatory sequence (IRS) 661 (1.4 µM) a specific blocking agent for endosomal TLR7 and not by a scrambled control nucleotide. ( C ) pDC are the source of Ab-RSV induced, TLR7 mediated production of IFN-α, as shown by intracellular staining for IFN-α in Lineage (CD3 neg. , CD14 neg. , CD19 neg. , CD16 neg. , CD56 neg. , Lin-1), MHC-II high , BDCA-4 + cells. The inhibitor brefeldin A was added at different time points post infection (the time points when BFA was added are given on the X-axis). Cytokines were allowed to accumulate for 10 hrs. after addition of BFA. ( D ) Purified pDC (obtained by negative selection removing CD3 + , CD19 + and CD16 + cells from fresh PBMC, followed by FACS purification of the BDCA-4 + cell population, which resulted in > 95% pure pDC) produce IFN-α upon infection with RSV. This response is abrogated after UV inactivation of RSV. In AS, both live RSV and UV-inactivated RSV induced IFN-α production to a similar extent. One representative experiment out of two performed with pDC isolated from two different donors is shown. ( E ) IFN-α production by Ab-RSV in purified pDC is blocked by IRS661 (1.4µM). ( F ) TLR1,-2 (PAM3CSK4, Peptidoglycan) and TLR4 (LPS) ligands suppress TLR9-triggered (ODN 2216) IFN-α production, but do not affect TLR7 (Gardiquimod) induced IFN-α production. All data represent mean ± SEM of triplicate measurements within 1 donor and analyzed using one way ANOVA followed by a Bonferroni post-test. ns not significant, *P
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    Image Search Results


    20A promotes autophagy induction through a mechanism that involves ATM activation. ( A ) HeLa cells were treated for the indicated times with 6 μM 20A . The activities of the MTORC1 and AMPK pathways were determined by immunoblot analysis using antibodies directed against phospho-4EBP1 (Thr37/46), 4EBP1, phospho-p70 (Thr 389), p70, phospho-AMPK (Thr 172) and AMPK. ( B ) Representative electron micrographs of HeLa cells untreated or treated for 16 h with 6 μM 20A . ( C ) Left, immunofluorescence analysis for the abundance of LC3 puncta (green dots) in HeLa cells after 3 h in either the absence or presence of 6 μM 20A ; Hoechst was used to stain nuclei (blue). Representative confocal images are shown; scale bar = 10 μm. Center, immunoblot images of LC3-II levels in HeLa cells treated with 6 μM 20A for the indicated times. Right, immunoblot of cells treated with or without E64d (10 μg/ml) plus pepstatin A (2 μg/ml) 2 h prior 20A treatment (6 μM, 8 h). ( D ) Immunoblot analysis of phospho-p62 (Ser 403) and p62 levels in HeLa cells treated with 6 μM 20A for the indicated times. ( E ) Immunoblot analysis of LC3-II levels in HeLa cells incubated with or without 10 μM KU55933 for 2 h and then treated with 6 μM 20A for 8 h. Where indicated, cells were incubated with 10 μg/ml E64d plus 2 μg/ml pepstatin A for 2 h prior 20A treatment. Where noted, bafilomycin A1 (50 nM) was added 3 h before cell lysis.

    Journal: Nucleic Acids Research

    Article Title: Modulation of the ATM/autophagy pathway by a G-quadruplex ligand tips the balance between senescence and apoptosis in cancer cells

    doi: 10.1093/nar/gkz095

    Figure Lengend Snippet: 20A promotes autophagy induction through a mechanism that involves ATM activation. ( A ) HeLa cells were treated for the indicated times with 6 μM 20A . The activities of the MTORC1 and AMPK pathways were determined by immunoblot analysis using antibodies directed against phospho-4EBP1 (Thr37/46), 4EBP1, phospho-p70 (Thr 389), p70, phospho-AMPK (Thr 172) and AMPK. ( B ) Representative electron micrographs of HeLa cells untreated or treated for 16 h with 6 μM 20A . ( C ) Left, immunofluorescence analysis for the abundance of LC3 puncta (green dots) in HeLa cells after 3 h in either the absence or presence of 6 μM 20A ; Hoechst was used to stain nuclei (blue). Representative confocal images are shown; scale bar = 10 μm. Center, immunoblot images of LC3-II levels in HeLa cells treated with 6 μM 20A for the indicated times. Right, immunoblot of cells treated with or without E64d (10 μg/ml) plus pepstatin A (2 μg/ml) 2 h prior 20A treatment (6 μM, 8 h). ( D ) Immunoblot analysis of phospho-p62 (Ser 403) and p62 levels in HeLa cells treated with 6 μM 20A for the indicated times. ( E ) Immunoblot analysis of LC3-II levels in HeLa cells incubated with or without 10 μM KU55933 for 2 h and then treated with 6 μM 20A for 8 h. Where indicated, cells were incubated with 10 μg/ml E64d plus 2 μg/ml pepstatin A for 2 h prior 20A treatment. Where noted, bafilomycin A1 (50 nM) was added 3 h before cell lysis.

    Article Snippet: Reagents ATM inhibitor KU-55933 (#SML1109), Hoechst 33258 (#14530), E64d (#516485), bafilomycin A1 ( # B1793), 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide, MTT (#M2128), blasticidine (#3513-03-9), Fluoromount (#F4680), puromycin (#58-58-2), Quinoline-Val-Asp-Difluorophenoxymethyl Ketone, QVD-OPH (# SML0063) and doxycycline (Dox) (#D9891) were purchased from Sigma-Aldrich.

    Techniques: Activation Assay, Immunofluorescence, Staining, Incubation, Lysis

    Identification of putative ion channel defective mutants by homology modeling and bafilomycin A1 rescue. A) Molecular models of N-terminal region mutants that yielded > 1 log reduction in infectious virus production compared to wild-type in a bicistronic context. The mutated residue Trp side chains are shown in red. Models provide insight into whether the mutation is likely to block the pore (e.g. H9W and S12W), disturb p7 intramolecular interactions (e.g. A10W), or interrupt p7 interactions with binding partners (e.g. A1W, A10W). B) Bafilomycin A1 rescue experiment schematic. Forty-eight hours post-electroporation, Huh-7.5 cells replicating control or p7 mutant viruses were supplied with cell culture medium containing bafilomycin A1 [8nM] or DMSO. Supernatants were collected 24 hours post-treatment, concentrated and dialyzed to remove excess bafilomycin A1, and then tittered on naïve Huh-7.5 cells to quantify infectious virus production. C) Resulting infectious virus titers from the experiment outlined in panel b. Mutant viruses yielding significantly more infectious virus production under bafilomycin A1 conditions compared with DMSO were identified using unpaired t-tests. Statistical results are indicated as follows: ns = not significant, * p

    Journal: PLoS Pathogens

    Article Title: The N-terminal Helical Region of the Hepatitis C Virus p7 Ion Channel Protein Is Critical for Infectious Virus Production

    doi: 10.1371/journal.ppat.1005297

    Figure Lengend Snippet: Identification of putative ion channel defective mutants by homology modeling and bafilomycin A1 rescue. A) Molecular models of N-terminal region mutants that yielded > 1 log reduction in infectious virus production compared to wild-type in a bicistronic context. The mutated residue Trp side chains are shown in red. Models provide insight into whether the mutation is likely to block the pore (e.g. H9W and S12W), disturb p7 intramolecular interactions (e.g. A10W), or interrupt p7 interactions with binding partners (e.g. A1W, A10W). B) Bafilomycin A1 rescue experiment schematic. Forty-eight hours post-electroporation, Huh-7.5 cells replicating control or p7 mutant viruses were supplied with cell culture medium containing bafilomycin A1 [8nM] or DMSO. Supernatants were collected 24 hours post-treatment, concentrated and dialyzed to remove excess bafilomycin A1, and then tittered on naïve Huh-7.5 cells to quantify infectious virus production. C) Resulting infectious virus titers from the experiment outlined in panel b. Mutant viruses yielding significantly more infectious virus production under bafilomycin A1 conditions compared with DMSO were identified using unpaired t-tests. Statistical results are indicated as follows: ns = not significant, * p

    Article Snippet: Bafilomycin A1 experiments To establish the bafilomycin concentration to be used in subsequent virus rescue experiments, bafilomycin A1 (Sigma Aldrich; or DMSO vehicle control) was titrated onto mock-electroporated cells and both viability and intracellular pH assessed 24 hrs post-treatment.

    Techniques: Mutagenesis, Blocking Assay, Binding Assay, Electroporation, Cell Culture

    ΔpH-driven 22 Na + uptake into glutamatergic synaptic vesicles is EIPA-sensitive Synaptic vesicle uptake of 22 Na + was measured for 10 minutes in ATP and either 10 mM choline glutamate or 10 mM choline aspartate ( a ), and the results normalized to uptake in glutamate (n=25). ( b ) 22 Na + uptake was measured in the presence of ATP, glutamate and either EIPA (15 μM), Bafilomycin A1 (Baf) (0.5 μM), Evans Blue (EB) (100 μM), (NH 4 ) 2 tartrate (NH + 4 ) (10 mM), or the Na + ionophore monensin (mon) (5 μM) as positive control (n=3–5). ( c ) Uptake in 10 mM choline glutamate or aspartate, with and without 2-aminoethoxydiphenylborate (2-APB) (50 μM), ruthenium red (RuR) (100 μM), tetraethylammonium (TEA) (5 mM), tetrodotoxin (TTX) (0.5 μM) and EIPA (50 μM) (n=3–5). ( d ) EIPA inhibits 22 Na + uptake more potently in vesicles acidified with glutamate (n=3–6). ( e ) Amiloride also inhibits 22 Na + uptake, but less potently than EIPA (n=5–6). *, p

    Journal: Nature neuroscience

    Article Title: Presynaptic Regulation of Quantal Size: K+/H+ Exchange Stimulates Glutamate Storage by Increasing Membrane Potential

    doi: 10.1038/nn.2898

    Figure Lengend Snippet: ΔpH-driven 22 Na + uptake into glutamatergic synaptic vesicles is EIPA-sensitive Synaptic vesicle uptake of 22 Na + was measured for 10 minutes in ATP and either 10 mM choline glutamate or 10 mM choline aspartate ( a ), and the results normalized to uptake in glutamate (n=25). ( b ) 22 Na + uptake was measured in the presence of ATP, glutamate and either EIPA (15 μM), Bafilomycin A1 (Baf) (0.5 μM), Evans Blue (EB) (100 μM), (NH 4 ) 2 tartrate (NH + 4 ) (10 mM), or the Na + ionophore monensin (mon) (5 μM) as positive control (n=3–5). ( c ) Uptake in 10 mM choline glutamate or aspartate, with and without 2-aminoethoxydiphenylborate (2-APB) (50 μM), ruthenium red (RuR) (100 μM), tetraethylammonium (TEA) (5 mM), tetrodotoxin (TTX) (0.5 μM) and EIPA (50 μM) (n=3–5). ( d ) EIPA inhibits 22 Na + uptake more potently in vesicles acidified with glutamate (n=3–6). ( e ) Amiloride also inhibits 22 Na + uptake, but less potently than EIPA (n=5–6). *, p

    Article Snippet: Materials Bafilomycin A1 was obtained from EMD Biosciences, Oxonol VI from Invitrogen, and other chemicals from Sigma.

    Techniques: Positive Control

    Entry mechanisms used by VSV-RV/CE2E1. Neutralization assay using goat anti-RV serum. VSV FLuc -RV/CE2E1 ( A ) and VSV FLuc -G ( B ) were incubated with the serially diluted goat antiserum against RV or with normal goat serum. Then, the Vero cells were infected with VSV FLuc -RV/CE2E1 or VSV FLuc -G pre-incubated with the serially diluted sera, and at 24 h p.i. the luciferase activities of the cells were measured. Data from VSV FLuc -RV/CE2E1 and VSV FLuc -G pre-incubated without sera were set to 100%. ( C , D ) Inhibition of VSV-RV/CE2E1 entry by lysomotrophic agents. Vero cells were treated with various concentrations of the following lysomotrophic agents: bafilomycin A1 ( C ), chloroquine ( D ), or were left untreated. The cells were infected with pseudotype VSVs bearing VSV, RV, MLV, or MV envelope proteins (VSV FLuc -G, VSV FLuc -RV/CE2E1, VSV FLuc -MLV/Env, and VSV FLuc -MV/FH, respectively). At 24 h p.i., the luciferase activities of the cells were measured. The luciferase activities of the cells not treated with lysomotrophic agents were set to 100%. ( E ) Ca 2+ dependency of VSV GFP -RV/CE2E1 infections. Vero cells were incubated with VSV GFP -RV/CE2E1 or VSV GFP -MV/FH at 4 °C for 2 h. After washing with PBS, the cells were incubated in DMEM containing various concentrations of CaCl 2 at 37 °C for 1 h. Next, the cells were incubated further for 24 h in standard DMEM containing 2 mM CaCl 2 , and the cell infectious units (CIUs) of the cells were determined. The CIU of the cells cultured continuously with 2 mM CaCl 2 was set to 100%. ( A – G ) Data represent the mean values ± SD for triplicate samples. ( A – D ) RLU, relative light unit of luciferase activity.

    Journal: Scientific Reports

    Article Title: Analysis of VSV pseudotype virus infection mediated by rubella virus envelope proteins

    doi: 10.1038/s41598-017-10865-2

    Figure Lengend Snippet: Entry mechanisms used by VSV-RV/CE2E1. Neutralization assay using goat anti-RV serum. VSV FLuc -RV/CE2E1 ( A ) and VSV FLuc -G ( B ) were incubated with the serially diluted goat antiserum against RV or with normal goat serum. Then, the Vero cells were infected with VSV FLuc -RV/CE2E1 or VSV FLuc -G pre-incubated with the serially diluted sera, and at 24 h p.i. the luciferase activities of the cells were measured. Data from VSV FLuc -RV/CE2E1 and VSV FLuc -G pre-incubated without sera were set to 100%. ( C , D ) Inhibition of VSV-RV/CE2E1 entry by lysomotrophic agents. Vero cells were treated with various concentrations of the following lysomotrophic agents: bafilomycin A1 ( C ), chloroquine ( D ), or were left untreated. The cells were infected with pseudotype VSVs bearing VSV, RV, MLV, or MV envelope proteins (VSV FLuc -G, VSV FLuc -RV/CE2E1, VSV FLuc -MLV/Env, and VSV FLuc -MV/FH, respectively). At 24 h p.i., the luciferase activities of the cells were measured. The luciferase activities of the cells not treated with lysomotrophic agents were set to 100%. ( E ) Ca 2+ dependency of VSV GFP -RV/CE2E1 infections. Vero cells were incubated with VSV GFP -RV/CE2E1 or VSV GFP -MV/FH at 4 °C for 2 h. After washing with PBS, the cells were incubated in DMEM containing various concentrations of CaCl 2 at 37 °C for 1 h. Next, the cells were incubated further for 24 h in standard DMEM containing 2 mM CaCl 2 , and the cell infectious units (CIUs) of the cells were determined. The CIU of the cells cultured continuously with 2 mM CaCl 2 was set to 100%. ( A – G ) Data represent the mean values ± SD for triplicate samples. ( A – D ) RLU, relative light unit of luciferase activity.

    Article Snippet: Chemicals Bafilomycin A1 and chloroquine were purchased from Sigma-Aldrich.

    Techniques: Neutralization, Incubation, Infection, Luciferase, Inhibition, Cell Culture, Activity Assay

    Deletion of ATG7 sensitizes cells to starvation without impacting proliferation under nutrient-replete conditions. ( A . ATG7-WT and ATG7-null clones 17 and 47 were treated for 24 h in the absence or presence of 50 ng/mL bafilomycin A1 and steady-state levels of endogenous markers visualized by Western blot. ( B ) Cells were cultured for 5 d in six-well dishes in regular growth medium (RPMI/FBS) or were starved for 3 d in HBSS followed by recovery for 2 d in regular growth medium. Cells then were fixed and stained with Crystal Violet. ( C ) Cells were grown in 96-well plates, and confluence was measured at the indicated time points by the IncuCyte imaging system. ( D ) A549 ATG7 wild-type or ATG7-null clonal cell lines generated by TALEN genomic editing were treated with or without 10 nM bafilomycin A1 for 24 h and then were lysed and assessed for modulation of ATG7 function. Vinculin was probed as a loading control. ( E ) A549 ATG7 wild-type and ATG7-null cell lines seeded in six-well dishes were maintained in regular growth medium (DMEM/FBS) or were subjected to starvation in HBSS for 5 d followed by recovery. Cells were stained with sulforhodamine B (SRB). ( F ) Equal cell numbers of A549 ATG7 wild-type or ATG7-null cells were plated, and proliferation was assessed after 4 d by SRB staining.

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

    Article Title: Macroautophagy is dispensable for growth of KRAS mutant tumors and chloroquine efficacy

    doi: 10.1073/pnas.1515617113

    Figure Lengend Snippet: Deletion of ATG7 sensitizes cells to starvation without impacting proliferation under nutrient-replete conditions. ( A . ATG7-WT and ATG7-null clones 17 and 47 were treated for 24 h in the absence or presence of 50 ng/mL bafilomycin A1 and steady-state levels of endogenous markers visualized by Western blot. ( B ) Cells were cultured for 5 d in six-well dishes in regular growth medium (RPMI/FBS) or were starved for 3 d in HBSS followed by recovery for 2 d in regular growth medium. Cells then were fixed and stained with Crystal Violet. ( C ) Cells were grown in 96-well plates, and confluence was measured at the indicated time points by the IncuCyte imaging system. ( D ) A549 ATG7 wild-type or ATG7-null clonal cell lines generated by TALEN genomic editing were treated with or without 10 nM bafilomycin A1 for 24 h and then were lysed and assessed for modulation of ATG7 function. Vinculin was probed as a loading control. ( E ) A549 ATG7 wild-type and ATG7-null cell lines seeded in six-well dishes were maintained in regular growth medium (DMEM/FBS) or were subjected to starvation in HBSS for 5 d followed by recovery. Cells were stained with sulforhodamine B (SRB). ( F ) Equal cell numbers of A549 ATG7 wild-type or ATG7-null cells were plated, and proliferation was assessed after 4 d by SRB staining.

    Article Snippet: The following chemicals were used: DMSO (Sigma), bafilomycin A1 (Tocris and EMD Millipore), chloroquine diphosphate (Tokyo Chemical Industry and Sigma), DOX (Clontech and Sigma), sulforhodamine B sodium salt (Sigma).

    Techniques: Clone Assay, Western Blot, Cell Culture, Staining, Imaging, Generated, Sulforhodamine B Assay

    Inhibition of autophagy with dominant-negative ATG4B. ( A ) Panc 10.05 cells with DOX-inducible expression of ATG4B C74A were cultured in the absence or presence of DOX for 5 d, and bafilomycin A1 was added for the last 24 h where indicated. Cells were lysed and probed by Western blot. ( B ) Panc 10.05 ATG4B C74A cells were grown for the indicated times with or without DOX, and steady-state levels of ATG4B, p62, LC3, and GAPDH were visualized by Western blot. ( C ) Panc 10.05 ATG4B C74A cells were cultured in 96-well plates with or without DOX, and cell growth was assessed using CellTiter-Glo at the indicated time points. Cell growth is depicted relative to day 0; data points represent the mean ± SD from two independent experiments.

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

    Article Title: Macroautophagy is dispensable for growth of KRAS mutant tumors and chloroquine efficacy

    doi: 10.1073/pnas.1515617113

    Figure Lengend Snippet: Inhibition of autophagy with dominant-negative ATG4B. ( A ) Panc 10.05 cells with DOX-inducible expression of ATG4B C74A were cultured in the absence or presence of DOX for 5 d, and bafilomycin A1 was added for the last 24 h where indicated. Cells were lysed and probed by Western blot. ( B ) Panc 10.05 ATG4B C74A cells were grown for the indicated times with or without DOX, and steady-state levels of ATG4B, p62, LC3, and GAPDH were visualized by Western blot. ( C ) Panc 10.05 ATG4B C74A cells were cultured in 96-well plates with or without DOX, and cell growth was assessed using CellTiter-Glo at the indicated time points. Cell growth is depicted relative to day 0; data points represent the mean ± SD from two independent experiments.

    Article Snippet: The following chemicals were used: DMSO (Sigma), bafilomycin A1 (Tocris and EMD Millipore), chloroquine diphosphate (Tokyo Chemical Industry and Sigma), DOX (Clontech and Sigma), sulforhodamine B sodium salt (Sigma).

    Techniques: Inhibition, Dominant Negative Mutation, Expressing, Cell Culture, Western Blot

    Effect of mAbs on levels of endogenous AQP4 in primary cultured astrocytes. (A) Representative immunoblots of lysates treated with 1 µg/ml of C9401 (lane 2), E5415A (lanes 3 and 4), or E5415B (lanes 5 and 6) in the absence (lanes 2, 3, and 5) or presence (lanes 4 and 6) of 500 nM bafilomycin A1 at 37 °C for 24 h using anti-AQP4 (upper panel) or anti-actin (lower panel) antibody. (B) Effect of E5415A or E5415B on levels of endogenous AQP4 (black column) and actin (grey column). Values are determined by Western blotting and estimated as fold relative to non-treated cells (A, lane 1). **( P

    Journal: Biochemistry and Biophysics Reports

    Article Title: The binding property of a monoclonal antibody against the extracellular domains of aquaporin-4 directs aquaporin-4 toward endocytosis

    doi: 10.1016/j.bbrep.2016.05.017

    Figure Lengend Snippet: Effect of mAbs on levels of endogenous AQP4 in primary cultured astrocytes. (A) Representative immunoblots of lysates treated with 1 µg/ml of C9401 (lane 2), E5415A (lanes 3 and 4), or E5415B (lanes 5 and 6) in the absence (lanes 2, 3, and 5) or presence (lanes 4 and 6) of 500 nM bafilomycin A1 at 37 °C for 24 h using anti-AQP4 (upper panel) or anti-actin (lower panel) antibody. (B) Effect of E5415A or E5415B on levels of endogenous AQP4 (black column) and actin (grey column). Values are determined by Western blotting and estimated as fold relative to non-treated cells (A, lane 1). **( P

    Article Snippet: To confirm lysosomal degradation of AQP4 in astrocytes, 500 nM bafilomycin A1 (Merck Millipore, Billerica, MA, USA) , was added to culture media and incubated for 24 h. Antibodies used were monoclonal anti-AQP4 (E5206, 1:2000, ref ); rabbit polyclonal anti-AQP4 (Sigma, St Louis, MO); and monoclonal anti-actin (2F3, 1:5000, Wako Pure Chemical Industries).

    Techniques: Cell Culture, Western Blot

    Model for the relief of BST-2-mediated restriction by Vpu. (A) Vpu recruits β-TrCP to induce ubiquitin-mediated trafficking events that remove BST-2 from the plasma membrane, its site of action as a virion-tethering factor. Circles in the cytoplasmic domain of Vpu represent phosphoserines 52 and 56. The interaction between BST-2 and Vpu and the ubiquitination of BST-2 are currently speculative. (B) Vpu induces bafilomycin A1-sensitive post-endocytic trafficking of BST-2 and endo-lysosomal degradation. The removal of BST-2 from the plasma membrane involves constitutive endocytosis of BST-2 via AP-2, followed by Vpu-mediated post-endocytic sorting events. Recycling of BST-2 to the plasma membrane in the absence of Vpu is currently speculative.

    Journal: PLoS Pathogens

    Article Title: Vpu Antagonizes BST-2-Mediated Restriction of HIV-1 Release via ?-TrCP and Endo-Lysosomal Trafficking

    doi: 10.1371/journal.ppat.1000450

    Figure Lengend Snippet: Model for the relief of BST-2-mediated restriction by Vpu. (A) Vpu recruits β-TrCP to induce ubiquitin-mediated trafficking events that remove BST-2 from the plasma membrane, its site of action as a virion-tethering factor. Circles in the cytoplasmic domain of Vpu represent phosphoserines 52 and 56. The interaction between BST-2 and Vpu and the ubiquitination of BST-2 are currently speculative. (B) Vpu induces bafilomycin A1-sensitive post-endocytic trafficking of BST-2 and endo-lysosomal degradation. The removal of BST-2 from the plasma membrane involves constitutive endocytosis of BST-2 via AP-2, followed by Vpu-mediated post-endocytic sorting events. Recycling of BST-2 to the plasma membrane in the absence of Vpu is currently speculative.

    Article Snippet: The vATPase inhibitor bafilomycin A1 was obtained from Sigma-Aldrich.

    Techniques:

    Bafilomycin A1 inhibits the ability of Vpu to down-regulate BST-2. (A) Cells (HeLa) were transfected with either an empty plasmid or a plasmid expressing Vpu, along with a plasmid expressing GFP as a transfection marker. Immediately after the transfection, the cells were treated with bafilomycin A1 (final concentration 0.13 µM in DMSO) or DMSO only for 14 hours, and then stained for surface or intracellular BST-2 and analyzed by two-color flow cytometry. Histograms represent the relative cell number vs. BST-2 fluorescence intensity for the GFP-positive cells. Gray-shaded histograms represent cells transfected to express Vpu; unshaded histograms represent cells transfected with the empty plasmid. The plots shown are representative of at least two transfections for each experimental condition. (B) Cells (HeLa) were transfected as described above. Immediately after the transfection, the cells were treated with bafilomycin A1 (final concentration 0.13 µM in DMSO), MG-132 (final concentration 30 µM in DMSO), or DMSO only for 14 hours, and then stained for surface BST-2 and analyzed by two-color flow cytometry. Histograms represent the relative cell number vs. BST-2 fluorescence intensity for the GFP-positive cells. Gray-shaded histograms represent cells transfected to express Vpu; unshaded histograms represent cells transfected with the empty plasmid. The plots shown are representative of two independent experiments.

    Journal: PLoS Pathogens

    Article Title: Vpu Antagonizes BST-2-Mediated Restriction of HIV-1 Release via ?-TrCP and Endo-Lysosomal Trafficking

    doi: 10.1371/journal.ppat.1000450

    Figure Lengend Snippet: Bafilomycin A1 inhibits the ability of Vpu to down-regulate BST-2. (A) Cells (HeLa) were transfected with either an empty plasmid or a plasmid expressing Vpu, along with a plasmid expressing GFP as a transfection marker. Immediately after the transfection, the cells were treated with bafilomycin A1 (final concentration 0.13 µM in DMSO) or DMSO only for 14 hours, and then stained for surface or intracellular BST-2 and analyzed by two-color flow cytometry. Histograms represent the relative cell number vs. BST-2 fluorescence intensity for the GFP-positive cells. Gray-shaded histograms represent cells transfected to express Vpu; unshaded histograms represent cells transfected with the empty plasmid. The plots shown are representative of at least two transfections for each experimental condition. (B) Cells (HeLa) were transfected as described above. Immediately after the transfection, the cells were treated with bafilomycin A1 (final concentration 0.13 µM in DMSO), MG-132 (final concentration 30 µM in DMSO), or DMSO only for 14 hours, and then stained for surface BST-2 and analyzed by two-color flow cytometry. Histograms represent the relative cell number vs. BST-2 fluorescence intensity for the GFP-positive cells. Gray-shaded histograms represent cells transfected to express Vpu; unshaded histograms represent cells transfected with the empty plasmid. The plots shown are representative of two independent experiments.

    Article Snippet: The vATPase inhibitor bafilomycin A1 was obtained from Sigma-Aldrich.

    Techniques: Transfection, Plasmid Preparation, Expressing, Marker, Concentration Assay, Staining, Flow Cytometry, Cytometry, Fluorescence

    TMR-r13 does not utilize the endocytic pathway to access the cytosolic space of cells. A, inhibitors of endocytic processes do not prevent cytosolic penetration. MCH58 cells were pretreated with 50 μ m amiloride, 200 n m bafilomycin, or PBS supplemented

    Journal: The Journal of Biological Chemistry

    Article Title: Membrane Oxidation Enables the Cytosolic Entry of Polyarginine Cell-penetrating Peptides *

    doi: 10.1074/jbc.M115.711564

    Figure Lengend Snippet: TMR-r13 does not utilize the endocytic pathway to access the cytosolic space of cells. A, inhibitors of endocytic processes do not prevent cytosolic penetration. MCH58 cells were pretreated with 50 μ m amiloride, 200 n m bafilomycin, or PBS supplemented

    Article Snippet: To inhibit endocytic processes, cells were first washed with warm PBS three times and pre-treated with 50 μ m amiloride (Sigma) or 200 n m bafilomycin A1 (Sigma) for 20 min. Alternatively, cells were washed with cold PBS and pre-incubated at 4 °C for 30 min. Then cells were incubated with 1 μ m TMR-r13 peptide in the presence of the same concentration of inhibitor (50 μ m amiloride or 200 n m bafilomycin A1) for 60 min at 37 °C or at 4 °C with TMR-r13 peptide alone for 60 min.

    Techniques:

    ARPE-19 cells were treated with 288 ng Resvega, 50 nM bafilomycin A1, or their combination for 12 h ( A ) in normal growth conditions and ( B ) a starvation-induced autophagy model. The protein level of p62 and LC3-I/LC3-II were analyzed by Western blot, while the expression was quantified in a comparison to α-tubulin and presented as a fold change compared to control. Western blotting data are shown as mean ± SD ( n = 3). * p

    Journal: Nutrients

    Article Title: Nutraceutical with Resveratrol and Omega-3 Fatty Acids Induces Autophagy in ARPE-19 Cells

    doi: 10.3390/nu8050284

    Figure Lengend Snippet: ARPE-19 cells were treated with 288 ng Resvega, 50 nM bafilomycin A1, or their combination for 12 h ( A ) in normal growth conditions and ( B ) a starvation-induced autophagy model. The protein level of p62 and LC3-I/LC3-II were analyzed by Western blot, while the expression was quantified in a comparison to α-tubulin and presented as a fold change compared to control. Western blotting data are shown as mean ± SD ( n = 3). * p

    Article Snippet: Treatments To study the effects of Resvega (Laboratoires Théa, Clermont-Ferrand, France) to induce autophagy, ARPE-19 cells were incubated in medium containing 288 ng Resvega corresponding to 25 µM of resveratrol (contents: 30 mg of trans resveratrol as a daily dose, 240 mg of vitamin C, 30 mg of vitamin E, 12.5 mg of zinc, 1 mg of copper, 665 mg of omega-3 fatty acids, 10 mg of lutein, and 2 mg of zeaxanthin), 1 µM proteasome inhibitor MG-132 (Calbiochem, Billerica, MA, USA), and 50 nM autophagy inhibitor bafilomycin A1 (Sigma-Aldrich, St. Louis, MO, USA).

    Techniques: Western Blot, Expressing

    Autophagy is activated during LSC’s stress response to UVA. (A) Representative images of autophagosomes in ATG7KD LSCs under UVA stress. Arrowheads show autophagic cells, asterisks indicate absence of autophagosomes. Scale bar, 50 μm. (B) Quantification of cells with autophagic activity in response to UVA. (C) Representative western blot image of autophagic flux in UVA-irradiated LSC colonies in absence and presence of BafA1, with or without UVA. LC3B-I and II were detected by immunoblotting at indicated time points. (D) Densitometric analysis of LC3B-II expression normalized to GAPDH. n = 3, * p

    Journal: PLoS ONE

    Article Title: Autophagy mediates cell cycle response by regulating nucleocytoplasmic transport of PAX6 in limbal stem cells under ultraviolet-A stress

    doi: 10.1371/journal.pone.0180868

    Figure Lengend Snippet: Autophagy is activated during LSC’s stress response to UVA. (A) Representative images of autophagosomes in ATG7KD LSCs under UVA stress. Arrowheads show autophagic cells, asterisks indicate absence of autophagosomes. Scale bar, 50 μm. (B) Quantification of cells with autophagic activity in response to UVA. (C) Representative western blot image of autophagic flux in UVA-irradiated LSC colonies in absence and presence of BafA1, with or without UVA. LC3B-I and II were detected by immunoblotting at indicated time points. (D) Densitometric analysis of LC3B-II expression normalized to GAPDH. n = 3, * p

    Article Snippet: LC3B-II expression in the absence or presence of autophagic flux inhibitor Bafilomycin A1 (BafA1, 100 nM from Streptomyces griseus , Sigma-Aldrich) with or without UVA irradiation was assessed at different time points up to 24 hours [ ].

    Techniques: Activity Assay, Western Blot, Irradiation, Expressing

    H 2 O 2 -induced oxidative stress impairs autophagic flux and decreases SIRT1 activity in H9C2 cells. H9C2 cells were treated with concentrations of H 2 O 2 for 12 hrs with or without bafilomycin A1 (Bafilo A1; 100 nM). Western blot analysis of protein levels of p62, cleaved caspase 3 (C-Caspase3) ( A ), SIRT1 ( C ), Ac-FOXO1, and total FOXO1 ( D ). Data are mean ± SEM. * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Resveratrol-enhanced autophagic flux ameliorates myocardial oxidative stress injury in diabetic mice

    doi: 10.1111/jcmm.12312

    Figure Lengend Snippet: H 2 O 2 -induced oxidative stress impairs autophagic flux and decreases SIRT1 activity in H9C2 cells. H9C2 cells were treated with concentrations of H 2 O 2 for 12 hrs with or without bafilomycin A1 (Bafilo A1; 100 nM). Western blot analysis of protein levels of p62, cleaved caspase 3 (C-Caspase3) ( A ), SIRT1 ( C ), Ac-FOXO1, and total FOXO1 ( D ). Data are mean ± SEM. * P

    Article Snippet: Mice were randomly divided into seven groups (n = 15 in each group) for treatment: Control, STZ, STZ+low-dose resveratrol (Sigma-Aldrich; STZ+RL), STZ+high-dose resveratrol (STZ+RH), STZ+RH+bafilomycin A1 (Sigma-Aldrich; STZ+RH+B), Control+ bafilomycin A1 (Control+B) and STZ+ bafilomycin A1 (STZ+B).

    Techniques: Activity Assay, Western Blot

    HCV transmission by exosomes and free virus can be blocked by proton pump inhibitor (Lansoprazole) and Vacuolar-type H+-ATPase inhibitor (bafilomycin A1). (A B) Huh 7.5 cells were pre-treated with Lansoprazole (2.5 µg/ml, 5 µg/ml, and 10 µg/ml) for 1 h at indicated concentrations, and then infected with (A) HCV exosomes or (B) cell free HCV virus for 24 h. Total protein was then extracted from cells and analyzed for HCV NS3 protein. (C D) Huh 7.5 cells were pre-treated with bafilomycin A1(Baf A1) (12.5 nM, 25 nM, 50 nM, and 100 nM) for one hour at concentrations indicated, then infected with (C) HCV J6/JFH-1 exosomes or (D) cell free HCV virus for 24 h. Total RNA was then extracted from cells and analyzed for HCV RNA. An MOI of 1 of HCV-exosomes and cell free HCV virus were used for all infections. Data presented here is representative of 3 independent experiments with p

    Journal: PLoS Pathogens

    Article Title: Exosomes from Hepatitis C Infected Patients Transmit HCV Infection and Contain Replication Competent Viral RNA in Complex with Ago2-miR122-HSP90

    doi: 10.1371/journal.ppat.1004424

    Figure Lengend Snippet: HCV transmission by exosomes and free virus can be blocked by proton pump inhibitor (Lansoprazole) and Vacuolar-type H+-ATPase inhibitor (bafilomycin A1). (A B) Huh 7.5 cells were pre-treated with Lansoprazole (2.5 µg/ml, 5 µg/ml, and 10 µg/ml) for 1 h at indicated concentrations, and then infected with (A) HCV exosomes or (B) cell free HCV virus for 24 h. Total protein was then extracted from cells and analyzed for HCV NS3 protein. (C D) Huh 7.5 cells were pre-treated with bafilomycin A1(Baf A1) (12.5 nM, 25 nM, 50 nM, and 100 nM) for one hour at concentrations indicated, then infected with (C) HCV J6/JFH-1 exosomes or (D) cell free HCV virus for 24 h. Total RNA was then extracted from cells and analyzed for HCV RNA. An MOI of 1 of HCV-exosomes and cell free HCV virus were used for all infections. Data presented here is representative of 3 independent experiments with p

    Article Snippet: Vacuolar-type H+-ATPase inhibitor (bafilomycin A1), proton pump inhibitor (Lansoprazole) and for inhibition of HCV Huh 7.5 cells derived exosomes and virus entry/replication Bafilomycin A1 was purchased from Sigma Aldrich and the proton pump inhibitor; Lansoprazole (Prevacid 24 hr OTC, Novartis), was purchased over the counter.

    Techniques: Transmission Assay, Infection

    Ece1-independent phagosome maturation. a IL-1β levels measured by ELISA in culture supernatants of LPS-primed hMDMs. Cells were treated with synthetic Candidalysin for 5 h. Selected samples were pre-treated with the vacuolar H+ ATPase inhibitor Bafilomycin A1 1 h prior to administration of synthetic Candidalysin. b , c Human MDMs were infected with C . albicans Wt, re-integrant ( ece1 Δ/Δ + ECE1 ) or mutant strains ( ece1 Δ/Δ, ece1 Δ/Δ + ECE1 Δ 184–279 ) (MOI 5) and co-localization of C . albicans -containing phagosomes with b the phagosomal marker LAMP1 or c the lysosomal acidification marker LysoTracker was quantified at indicated time points. d Human MDMs pre-stained with LysoTracker were infected with heat-killed C . albicans Wt (MOI 5) for 3 h in presence or absence of Bafilomycin A1 (phagosomal acidification inhibitor) or synthetic Candidalysin. C . albicans -containing phagosomes were quantified for the percentage of LysoTracker-positive phagosomes and LysoTracker intensity. e Murine RAW264.7 Dectin-1 macrophages were infected with C . albicans Wt or ece1 Δ/Δ mutant yeasts (MOI 2) and co-localization of C . albicans -containing phagosomes with the phago(lyso)somal markers Lamp1, PI(4)P, and Rab7 was quantified at indicated time points. f , g Murine RAW264.7 Dectin-1 macrophages were infected with C . albicans Wt or ece1 Δ/Δ mutant strain as described in e . Representative image of Lamp1 ( f ) or PI(4)P and Rab7 ( g ) acquisition 30 min after phagocytosis. ConA staining of non-phagocytosed C . albicans . Scale bar 8 μm. Values are represented as scatterplot with median of three independent donors or experiments ( n ≥ 3)

    Journal: Nature Communications

    Article Title: The fungal peptide toxin Candidalysin activates the NLRP3 inflammasome and causes cytolysis in mononuclear phagocytes

    doi: 10.1038/s41467-018-06607-1

    Figure Lengend Snippet: Ece1-independent phagosome maturation. a IL-1β levels measured by ELISA in culture supernatants of LPS-primed hMDMs. Cells were treated with synthetic Candidalysin for 5 h. Selected samples were pre-treated with the vacuolar H+ ATPase inhibitor Bafilomycin A1 1 h prior to administration of synthetic Candidalysin. b , c Human MDMs were infected with C . albicans Wt, re-integrant ( ece1 Δ/Δ + ECE1 ) or mutant strains ( ece1 Δ/Δ, ece1 Δ/Δ + ECE1 Δ 184–279 ) (MOI 5) and co-localization of C . albicans -containing phagosomes with b the phagosomal marker LAMP1 or c the lysosomal acidification marker LysoTracker was quantified at indicated time points. d Human MDMs pre-stained with LysoTracker were infected with heat-killed C . albicans Wt (MOI 5) for 3 h in presence or absence of Bafilomycin A1 (phagosomal acidification inhibitor) or synthetic Candidalysin. C . albicans -containing phagosomes were quantified for the percentage of LysoTracker-positive phagosomes and LysoTracker intensity. e Murine RAW264.7 Dectin-1 macrophages were infected with C . albicans Wt or ece1 Δ/Δ mutant yeasts (MOI 2) and co-localization of C . albicans -containing phagosomes with the phago(lyso)somal markers Lamp1, PI(4)P, and Rab7 was quantified at indicated time points. f , g Murine RAW264.7 Dectin-1 macrophages were infected with C . albicans Wt or ece1 Δ/Δ mutant strain as described in e . Representative image of Lamp1 ( f ) or PI(4)P and Rab7 ( g ) acquisition 30 min after phagocytosis. ConA staining of non-phagocytosed C . albicans . Scale bar 8 μm. Values are represented as scatterplot with median of three independent donors or experiments ( n ≥ 3)

    Article Snippet: For inhibitor studies, the following compounds were added 1 h prior to infection: the caspase-1-inhibitor Z-YVAD-FMK (88.9 µM; Merck) or Ac-YVAD-cmk (20 µM, Invivogen), the caspase-1-inhibitor VX-765 (50 µg/mL, Invivogen) or vehicle control, the actin cytoskeleton inhibitor Cytochalasin D (10 µM; Sigma Aldrich), the V-ATPase inhibitor Bafilomycin A1 (50–500 nM; Sigma Aldrich), the ROS inhibitor 4-Aminopyrrolidine-2,4-dicarboxylate (PDTC) (100, 500 µM; Enzo Life Sciences), the potassium channel inhibitor glibenclamide (25 µM; Sigma Aldrich) or the RIP1-kinase inhibitor Necrostatin-1 (12.5–50 µM; Biomol).

    Techniques: Enzyme-linked Immunosorbent Assay, Infection, Mutagenesis, Marker, Staining

    Effects of collagen α-5(IV) chain (Col4a5) knockdown on the V-ATPase activity and subunit expression in Raw264.7 macrophages. (A) Relative microsomal V-ATPase activity in control (VC) and Col4a5-knockdown (C51, C52 and C53) macrophage cell lines. (B) Colony forming units (CFUs) of mycobacterium bovis Calmette-Guérin ( BCG ) were counted in VC, C51, C52 and C53 cell lines infected with BCG at MOI 3, which were treated with the V-ATPase-specific inhibitor bafilomycin A1 (BA1) or in controls (Con). (C) Western blot analysis detected protein bands of the V-ATPase subunits B (VAB) and E (VAE) in VC, C51, C52 and C53 cell lines without BCG infection (MOI 0) or with BCG infection (MOI 2, 8). Tubulin served as loading control. * P

    Journal: International Journal of Molecular Medicine

    Article Title: A potential target gene for the host-directed therapy of mycobacterial infection in murine macrophages

    doi: 10.3892/ijmm.2016.2675

    Figure Lengend Snippet: Effects of collagen α-5(IV) chain (Col4a5) knockdown on the V-ATPase activity and subunit expression in Raw264.7 macrophages. (A) Relative microsomal V-ATPase activity in control (VC) and Col4a5-knockdown (C51, C52 and C53) macrophage cell lines. (B) Colony forming units (CFUs) of mycobacterium bovis Calmette-Guérin ( BCG ) were counted in VC, C51, C52 and C53 cell lines infected with BCG at MOI 3, which were treated with the V-ATPase-specific inhibitor bafilomycin A1 (BA1) or in controls (Con). (C) Western blot analysis detected protein bands of the V-ATPase subunits B (VAB) and E (VAE) in VC, C51, C52 and C53 cell lines without BCG infection (MOI 0) or with BCG infection (MOI 2, 8). Tubulin served as loading control. * P

    Article Snippet: Reactions with and without the V-ATPase-specific inhibitor bafilomycin A1 (BA1; 100 nM; Sigma-Aldrich) were started by the addition of ATP to a final concentration of 2 mM in a microtiter plate, and absorbance at 340 nm was monitored for 20 min in a Safire plate reader (Tecan).

    Techniques: Activity Assay, Expressing, Infection, Western Blot

    The stimulation-induced alkalinizing phase is selectively blocked by inhibiting exocytosis (A) and by inhibiting vesicular H + -ATPase (vATPase, B). A, Responses before and after exposure to 20 nM BoNT A (left) or 14.5 nM BoNT E (right) for 80-140 min. B, Responses before and after exposure to 1 μM folimycin (left) or 1 μM bafilomycin (right) for 1-4 hr. Folimycin produced no significant change in resting fluorescence. Ca, averaged effects of these drugs and of vesamicol on the magnitudes of the early acidifying response (measured 3-4 s following stimulation onset) and the late alkalinizing response (measured 3-4 s following cessation of stimulation). Vesamicol (7 μM) exposure was 3 hr (n=4 terminals). Results with both BoNTs were averaged together, as were results with foli- and bafilo-mycin (fol/baf). * indicates significant difference from control (p

    Journal: Neuron

    Article Title: Vesicular ATPase inserted into the plasma membrane of motor terminals by exocytosis alkalinizes cytosolic pH and facilitates endocytosis

    doi: 10.1016/j.neuron.2010.11.035

    Figure Lengend Snippet: The stimulation-induced alkalinizing phase is selectively blocked by inhibiting exocytosis (A) and by inhibiting vesicular H + -ATPase (vATPase, B). A, Responses before and after exposure to 20 nM BoNT A (left) or 14.5 nM BoNT E (right) for 80-140 min. B, Responses before and after exposure to 1 μM folimycin (left) or 1 μM bafilomycin (right) for 1-4 hr. Folimycin produced no significant change in resting fluorescence. Ca, averaged effects of these drugs and of vesamicol on the magnitudes of the early acidifying response (measured 3-4 s following stimulation onset) and the late alkalinizing response (measured 3-4 s following cessation of stimulation). Vesamicol (7 μM) exposure was 3 hr (n=4 terminals). Results with both BoNTs were averaged together, as were results with foli- and bafilo-mycin (fol/baf). * indicates significant difference from control (p

    Article Snippet: Reagent sources: agatoxin (Alomone Labs, Jerusalem, Israel); botulinum toxins (BBTech, North Dartmouth, MA); foli/bafilo-mycin (EMD Chemicals, Gibbstown, NJ); Oregon Green (OG)-1, FM1-43fx (Invitrogen, Carlsbad, CA); dynasore (Sigma Aldrich).

    Techniques: Produced, Fluorescence

    Effects of Baf A1, a pharmacological inhibitor of virus uncoating in endosomes, on cytokine and chemokine production by primary microglia cultures prepared from wild-type mice and then exposed to NSV Some wells were treated with Baf A1 as outlined in Materials and Methods section. A minimum of three wells either without or with Baf A1 pretreatment were then exposed to NSV, and cytokine and chemokine concentrations measured by ELISA in culture supernatants 24 h later using ELISA-specific for IL-12p40 ( A ), IFN-α ( B ), CCL2 ( C ) and CCL5 ( D ). The production of these mediators without or with Baf A1 treatment was measured using primary cells prepared on two separate occasions.

    Journal: ASN NEURO

    Article Title: Complexity of the microglial activation pathways that drive innate host responses during lethal alphavirus encephalitis in mice

    doi: 10.1042/AN20120016

    Figure Lengend Snippet: Effects of Baf A1, a pharmacological inhibitor of virus uncoating in endosomes, on cytokine and chemokine production by primary microglia cultures prepared from wild-type mice and then exposed to NSV Some wells were treated with Baf A1 as outlined in Materials and Methods section. A minimum of three wells either without or with Baf A1 pretreatment were then exposed to NSV, and cytokine and chemokine concentrations measured by ELISA in culture supernatants 24 h later using ELISA-specific for IL-12p40 ( A ), IFN-α ( B ), CCL2 ( C ) and CCL5 ( D ). The production of these mediators without or with Baf A1 treatment was measured using primary cells prepared on two separate occasions.

    Article Snippet: Some experiments were conducted in the presence of 20 nM of Baf A1 (bafilomycin A1) (Sigma–Aldrich) to prevent the acidification of endosomes and thus blocking virus uncoating by inhibiting acid-induced fusion of viral envelopes and the endosomal membrane ( ).

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay

    CD14 + monocytes inhibit IFN-α production triggered by Ab-RSV via TLR7 in pDC. ( A ) IFN-α production in CD14 + cell depleted PBMCs induced by Ab-RSV complexes, TLR9 ligand ODN 2216 and TLR7 ligand imiquimod is decreased by blocking endosomal acidification with 50nM Bafilomycin A 1 . ( B ) Ab-RSV-induced IFN-α production in CD14 + cell depleted PBMCs was abrogated in the presence of immune regulatory sequence (IRS) 661 (1.4 µM) a specific blocking agent for endosomal TLR7 and not by a scrambled control nucleotide. ( C ) pDC are the source of Ab-RSV induced, TLR7 mediated production of IFN-α, as shown by intracellular staining for IFN-α in Lineage (CD3 neg. , CD14 neg. , CD19 neg. , CD16 neg. , CD56 neg. , Lin-1), MHC-II high , BDCA-4 + cells. The inhibitor brefeldin A was added at different time points post infection (the time points when BFA was added are given on the X-axis). Cytokines were allowed to accumulate for 10 hrs. after addition of BFA. ( D ) Purified pDC (obtained by negative selection removing CD3 + , CD19 + and CD16 + cells from fresh PBMC, followed by FACS purification of the BDCA-4 + cell population, which resulted in > 95% pure pDC) produce IFN-α upon infection with RSV. This response is abrogated after UV inactivation of RSV. In AS, both live RSV and UV-inactivated RSV induced IFN-α production to a similar extent. One representative experiment out of two performed with pDC isolated from two different donors is shown. ( E ) IFN-α production by Ab-RSV in purified pDC is blocked by IRS661 (1.4µM). ( F ) TLR1,-2 (PAM3CSK4, Peptidoglycan) and TLR4 (LPS) ligands suppress TLR9-triggered (ODN 2216) IFN-α production, but do not affect TLR7 (Gardiquimod) induced IFN-α production. All data represent mean ± SEM of triplicate measurements within 1 donor and analyzed using one way ANOVA followed by a Bonferroni post-test. ns not significant, *P

    Journal: PLoS ONE

    Article Title: Respiratory Syncytial Virus Induced Type I IFN Production by pDC Is Regulated by RSV-Infected Airway Epithelial Cells, RSV-Exposed Monocytes and Virus Specific Antibodies

    doi: 10.1371/journal.pone.0081695

    Figure Lengend Snippet: CD14 + monocytes inhibit IFN-α production triggered by Ab-RSV via TLR7 in pDC. ( A ) IFN-α production in CD14 + cell depleted PBMCs induced by Ab-RSV complexes, TLR9 ligand ODN 2216 and TLR7 ligand imiquimod is decreased by blocking endosomal acidification with 50nM Bafilomycin A 1 . ( B ) Ab-RSV-induced IFN-α production in CD14 + cell depleted PBMCs was abrogated in the presence of immune regulatory sequence (IRS) 661 (1.4 µM) a specific blocking agent for endosomal TLR7 and not by a scrambled control nucleotide. ( C ) pDC are the source of Ab-RSV induced, TLR7 mediated production of IFN-α, as shown by intracellular staining for IFN-α in Lineage (CD3 neg. , CD14 neg. , CD19 neg. , CD16 neg. , CD56 neg. , Lin-1), MHC-II high , BDCA-4 + cells. The inhibitor brefeldin A was added at different time points post infection (the time points when BFA was added are given on the X-axis). Cytokines were allowed to accumulate for 10 hrs. after addition of BFA. ( D ) Purified pDC (obtained by negative selection removing CD3 + , CD19 + and CD16 + cells from fresh PBMC, followed by FACS purification of the BDCA-4 + cell population, which resulted in > 95% pure pDC) produce IFN-α upon infection with RSV. This response is abrogated after UV inactivation of RSV. In AS, both live RSV and UV-inactivated RSV induced IFN-α production to a similar extent. One representative experiment out of two performed with pDC isolated from two different donors is shown. ( E ) IFN-α production by Ab-RSV in purified pDC is blocked by IRS661 (1.4µM). ( F ) TLR1,-2 (PAM3CSK4, Peptidoglycan) and TLR4 (LPS) ligands suppress TLR9-triggered (ODN 2216) IFN-α production, but do not affect TLR7 (Gardiquimod) induced IFN-α production. All data represent mean ± SEM of triplicate measurements within 1 donor and analyzed using one way ANOVA followed by a Bonferroni post-test. ns not significant, *P

    Article Snippet: Inhibition assays To determine the involvement of specific innate immune pathways we employed inhibitors to block endosomal acidification (Bafilomycin A1 , 50nM, Calbiochem) and protein transport (Brefeldin A, 40nM, Sigma-Aldrich).

    Techniques: Blocking Assay, Sequencing, Staining, Infection, Purification, Selection, FACS, Isolation