filipin Thermo Fisher Search Results


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  • 99
    Thermo Fisher ampflstr identifiler pcr amplification kit
    Detection of genetic changes in post-cell fusion cells and a proposed model of cell fusion in tumor progression (a). Ten single cell clones including 2 pS-puro-scrambled (clones 1-2), 2 pEYFP-N3-neo (clones 3-4), and 6 pS-puro-scrambled-pEYFP-N3-neo double selected clones (clones 5-10) were selected at random and amplified in cell culture. Genomic DNA was extracted from the cells. AmpFlSTR <t>Identifiler</t> <t>PCR</t> Amplification kit was used to examine microsatellite markers. Shown are fragment analysis of the D19S433 alleles. Note the fragment height of allele a (in the red box) was decreased in the fusion cells (6-9), and lost completely in clone #10 (b). A proposed model of K type human endogenous retrovirus in mediating intercellular fusion, evolution of genetic changes, and malignant progression
    Ampflstr Identifiler Pcr Amplification Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1426 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore filipin
    Ezetimibe is protective in a zebrafish model of S . Typhi infection. ( A ) Fish infected with S . Typhi prgH had increased survival compared with fish infected with WT S . Typhi ( P = 0.01). The survival curve was carried out for 5 d; fish were checked once each day. ( B ) Zebrafish were scored 24 h post S . Typhi infection as cleared (no bacteria), localized (bacteria only in the swim bladder), disseminated (bacteria found outside the swim bladder), or dead (fish dead due to bacterial burden). The swim bladders are denoted by red circles; bacteria are denoted by the red arrows. ( C ) Fish infected with the S . Typhi prgH mutant had increased clearance of bacteria at 24 h ( P = 0.03). ( D ) Ezetimibe had no effect on S . Typhi bacterial growth. Bacteria were diluted from an overnight stock and grown with DMSO or 10 µM ezetimibe. The OD 600 was taken every 30 min for 3.5 h. Data points are the mean from two separate experiments. ( E ) Ezetimibe decreased <t>filipin</t> staining in fish. Twenty-four–hour pretreatment with 10 µM ezetimibe reduced filipin (0.05 mg/mL) staining ( P = 0.003) in zebrafish; n = 20 fish from two separate experiments; P value from an unpaired t test. ( F ) Fish pretreated with ezetimibe had increased survival from S . Typhi infection compared with DMSO-pretreated controls ( P = 0.03). ( G ) Ezetimibe treatment increased bacterial clearance in fish. Twenty-four–hour pretreatment with 10 µM ezetimibe increased the percentage of fish that cleared the bacteria 24 h postinfection from 8 to 30% ( P = 0.009). Infection data for each survival curve and clearance comparisons are from three independent experiments with a minimum of n = 60 fish. P values from survival curves are from the Mantel–Cox test; P values for other comparisons are from unpaired t tests.
    Filipin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2595 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher phosphate buffered saline
    Ezetimibe is protective in a zebrafish model of S . Typhi infection. ( A ) Fish infected with S . Typhi prgH had increased survival compared with fish infected with WT S . Typhi ( P = 0.01). The survival curve was carried out for 5 d; fish were checked once each day. ( B ) Zebrafish were scored 24 h post S . Typhi infection as cleared (no bacteria), localized (bacteria only in the swim bladder), disseminated (bacteria found outside the swim bladder), or dead (fish dead due to bacterial burden). The swim bladders are denoted by red circles; bacteria are denoted by the red arrows. ( C ) Fish infected with the S . Typhi prgH mutant had increased clearance of bacteria at 24 h ( P = 0.03). ( D ) Ezetimibe had no effect on S . Typhi bacterial growth. Bacteria were diluted from an overnight stock and grown with DMSO or 10 µM ezetimibe. The OD 600 was taken every 30 min for 3.5 h. Data points are the mean from two separate experiments. ( E ) Ezetimibe decreased <t>filipin</t> staining in fish. Twenty-four–hour pretreatment with 10 µM ezetimibe reduced filipin (0.05 mg/mL) staining ( P = 0.003) in zebrafish; n = 20 fish from two separate experiments; P value from an unpaired t test. ( F ) Fish pretreated with ezetimibe had increased survival from S . Typhi infection compared with DMSO-pretreated controls ( P = 0.03). ( G ) Ezetimibe treatment increased bacterial clearance in fish. Twenty-four–hour pretreatment with 10 µM ezetimibe increased the percentage of fish that cleared the bacteria 24 h postinfection from 8 to 30% ( P = 0.009). Infection data for each survival curve and clearance comparisons are from three independent experiments with a minimum of n = 60 fish. P values from survival curves are from the Mantel–Cox test; P values for other comparisons are from unpaired t tests.
    Phosphate Buffered Saline, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 34842 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher hoechst 33342
    slarp (-) parasites are impaired in liver stage development. (A) HepG2 cells were infected with WT or slarp (-) sporozoites (1×10 4 ) and cultured for 6–72 hours before staining with anti-CSP or anti-HSP70 antibodies and quantification by fluorescence microscopy. Results are expressed as the mean number of parasites from triplicate wells+/−SD. DC, detached infected cells. (B) HepG2 cells were infected with WT or slarp(-) sporozoites and cultured for 24, 48 or 72 hours before staining with anti-HSP70 antibodies (green) and <t>Hoechst</t> 33342 (blue), and examination by confocal fluorescence microscopy. Bar = 10 µm. (C) HepG2 cells were infected with WT or slarp(-) sporozoites and cultured for 24, 48 or 72 hours before staining with anti-HSP70 antibodies and Hoechst 33342. For each population, the number of nuclei in at least 50 parasites was determined by examination under a fluorescence microscope.
    Hoechst 33342, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 54383 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher dapi
    Intracellular localization of the Arg-nHAP/DZ1 complex in CNE1-LMP1 cells. Cells were fixed with 4% paraformaldehyde at 4°c for 30 minutes. Nuclei were stained with <t>DAPI</t> (200×). Abbreviations: Arg-nHAP, arginine-modified nanohydroxyapatite particles; DAPI, 4′,6-diamidino-2-phenylindole; DZ1, DNAzyme 1; LF2000, <t>lipofectamine</t> 2000; FITC, fluorescein isothiocyanate.
    Dapi, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 155801 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher amplex red cholesterol assay kit
    Intracellular localization of the Arg-nHAP/DZ1 complex in CNE1-LMP1 cells. Cells were fixed with 4% paraformaldehyde at 4°c for 30 minutes. Nuclei were stained with <t>DAPI</t> (200×). Abbreviations: Arg-nHAP, arginine-modified nanohydroxyapatite particles; DAPI, 4′,6-diamidino-2-phenylindole; DZ1, DNAzyme 1; LF2000, <t>lipofectamine</t> 2000; FITC, fluorescein isothiocyanate.
    Amplex Red Cholesterol Assay Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 2553 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher lipofectamine 2000
    Intracellular localization of the Arg-nHAP/DZ1 complex in CNE1-LMP1 cells. Cells were fixed with 4% paraformaldehyde at 4°c for 30 minutes. Nuclei were stained with DAPI (200×). Abbreviations:  Arg-nHAP, arginine-modified nanohydroxyapatite particles; DAPI, 4′,6-diamidino-2-phenylindole; DZ1, DNAzyme 1; LF2000, lipofectamine 2000; FITC, fluorescein isothiocyanate.
    Lipofectamine 2000, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 466214 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher alexa fluor 488
    Confocal microscopy and adherence-invasion assays with pharmacologic inhibitors. Lipid raft-independent invasion of bronchial epithelial cells by NTHI. (A) Confocal microscopy. At 4 and 24 h postinoculation of H292 cells, NTHI does not colocalize with vesicles positive for the following markers of lipid rafts: caveolin-1 (shown at 24 h), flotillin-1 (shown at 24 h), and cholesterol (shown at 4 h). NTHI was labeled with anti-11P6H antibody conjugated to <t>Alexa</t> <t>Fluor</t> 488 (green). Caveolin-1 was labeled with anti-caveolin-1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). Flotillin-1 was labeled with anti-flotillin-1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). Cholesterol was labeled with filipin (blue), and those samples were also labeled with anti-human secretory component antibody and donkey anti-goat antibody conjugated to Alexa Fluor 568 (red) to provide additional visualization of the plasma membrane. (B) Confocal microscopy. Filipin (FLP) and nystatin (NST) inhibit the internalization of fluorescently conjugated cholera toxin B subunit (white arrows), a known cargo of lipid raft-mediated endocytosis. H292 cells were pretreated with sRPMI containing inhibitor or inhibitor diluent, followed by addition of the cargo conjugate, incubation for 20 min on ice, and incubation for 2 h at 37°C. (C) Adherence-invasion assays. Filipin and nystatin do not inhibit invasion by NTHI. H292 cells were pretreated with inhibitor or inhibitor diluent for 2 h, followed by a 4-h infection conducted according to the adherence-invasion assay protocol. Data are normalized to samples in the absence of inhibitor. Error bars represent standard errors of the means of three independent experiments. A paired Student t test was used to calculate statistical significance. *, P ≤ 0.05; **, P ≤ 0.005.
    Alexa Fluor 488, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 73927 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher bsa
    Confocal microscopy and adherence-invasion assays with pharmacologic inhibitors. Lipid raft-independent invasion of bronchial epithelial cells by NTHI. (A) Confocal microscopy. At 4 and 24 h postinoculation of H292 cells, NTHI does not colocalize with vesicles positive for the following markers of lipid rafts: caveolin-1 (shown at 24 h), flotillin-1 (shown at 24 h), and cholesterol (shown at 4 h). NTHI was labeled with anti-11P6H antibody conjugated to <t>Alexa</t> <t>Fluor</t> 488 (green). Caveolin-1 was labeled with anti-caveolin-1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). Flotillin-1 was labeled with anti-flotillin-1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). Cholesterol was labeled with filipin (blue), and those samples were also labeled with anti-human secretory component antibody and donkey anti-goat antibody conjugated to Alexa Fluor 568 (red) to provide additional visualization of the plasma membrane. (B) Confocal microscopy. Filipin (FLP) and nystatin (NST) inhibit the internalization of fluorescently conjugated cholera toxin B subunit (white arrows), a known cargo of lipid raft-mediated endocytosis. H292 cells were pretreated with sRPMI containing inhibitor or inhibitor diluent, followed by addition of the cargo conjugate, incubation for 20 min on ice, and incubation for 2 h at 37°C. (C) Adherence-invasion assays. Filipin and nystatin do not inhibit invasion by NTHI. H292 cells were pretreated with inhibitor or inhibitor diluent for 2 h, followed by a 4-h infection conducted according to the adherence-invasion assay protocol. Data are normalized to samples in the absence of inhibitor. Error bars represent standard errors of the means of three independent experiments. A paired Student t test was used to calculate statistical significance. *, P ≤ 0.05; **, P ≤ 0.005.
    Bsa, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 25730 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher fetal bovine serum
    Confocal microscopy and adherence-invasion assays with pharmacologic inhibitors. Lipid raft-independent invasion of bronchial epithelial cells by NTHI. (A) Confocal microscopy. At 4 and 24 h postinoculation of H292 cells, NTHI does not colocalize with vesicles positive for the following markers of lipid rafts: caveolin-1 (shown at 24 h), flotillin-1 (shown at 24 h), and cholesterol (shown at 4 h). NTHI was labeled with anti-11P6H antibody conjugated to <t>Alexa</t> <t>Fluor</t> 488 (green). Caveolin-1 was labeled with anti-caveolin-1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). Flotillin-1 was labeled with anti-flotillin-1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). Cholesterol was labeled with filipin (blue), and those samples were also labeled with anti-human secretory component antibody and donkey anti-goat antibody conjugated to Alexa Fluor 568 (red) to provide additional visualization of the plasma membrane. (B) Confocal microscopy. Filipin (FLP) and nystatin (NST) inhibit the internalization of fluorescently conjugated cholera toxin B subunit (white arrows), a known cargo of lipid raft-mediated endocytosis. H292 cells were pretreated with sRPMI containing inhibitor or inhibitor diluent, followed by addition of the cargo conjugate, incubation for 20 min on ice, and incubation for 2 h at 37°C. (C) Adherence-invasion assays. Filipin and nystatin do not inhibit invasion by NTHI. H292 cells were pretreated with inhibitor or inhibitor diluent for 2 h, followed by a 4-h infection conducted according to the adherence-invasion assay protocol. Data are normalized to samples in the absence of inhibitor. Error bars represent standard errors of the means of three independent experiments. A paired Student t test was used to calculate statistical significance. *, P ≤ 0.05; **, P ≤ 0.005.
    Fetal Bovine Serum, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 240932 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher lysotracker red dnd 99
    Confocal microscopy and adherence-invasion assays with pharmacologic inhibitors. Lipid raft-independent invasion of bronchial epithelial cells by NTHI. (A) Confocal microscopy. At 4 and 24 h postinoculation of H292 cells, NTHI does not colocalize with vesicles positive for the following markers of lipid rafts: caveolin-1 (shown at 24 h), flotillin-1 (shown at 24 h), and cholesterol (shown at 4 h). NTHI was labeled with anti-11P6H antibody conjugated to <t>Alexa</t> <t>Fluor</t> 488 (green). Caveolin-1 was labeled with anti-caveolin-1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). Flotillin-1 was labeled with anti-flotillin-1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). Cholesterol was labeled with filipin (blue), and those samples were also labeled with anti-human secretory component antibody and donkey anti-goat antibody conjugated to Alexa Fluor 568 (red) to provide additional visualization of the plasma membrane. (B) Confocal microscopy. Filipin (FLP) and nystatin (NST) inhibit the internalization of fluorescently conjugated cholera toxin B subunit (white arrows), a known cargo of lipid raft-mediated endocytosis. H292 cells were pretreated with sRPMI containing inhibitor or inhibitor diluent, followed by addition of the cargo conjugate, incubation for 20 min on ice, and incubation for 2 h at 37°C. (C) Adherence-invasion assays. Filipin and nystatin do not inhibit invasion by NTHI. H292 cells were pretreated with inhibitor or inhibitor diluent for 2 h, followed by a 4-h infection conducted according to the adherence-invasion assay protocol. Data are normalized to samples in the absence of inhibitor. Error bars represent standard errors of the means of three independent experiments. A paired Student t test was used to calculate statistical significance. *, P ≤ 0.05; **, P ≤ 0.005.
    Lysotracker Red Dnd 99, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 5869 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher prolong gold antifade reagent
    Confocal microscopy and adherence-invasion assays with pharmacologic inhibitors. Lipid raft-independent invasion of bronchial epithelial cells by NTHI. (A) Confocal microscopy. At 4 and 24 h postinoculation of H292 cells, NTHI does not colocalize with vesicles positive for the following markers of lipid rafts: caveolin-1 (shown at 24 h), flotillin-1 (shown at 24 h), and cholesterol (shown at 4 h). NTHI was labeled with anti-11P6H antibody conjugated to <t>Alexa</t> <t>Fluor</t> 488 (green). Caveolin-1 was labeled with anti-caveolin-1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). Flotillin-1 was labeled with anti-flotillin-1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). Cholesterol was labeled with filipin (blue), and those samples were also labeled with anti-human secretory component antibody and donkey anti-goat antibody conjugated to Alexa Fluor 568 (red) to provide additional visualization of the plasma membrane. (B) Confocal microscopy. Filipin (FLP) and nystatin (NST) inhibit the internalization of fluorescently conjugated cholera toxin B subunit (white arrows), a known cargo of lipid raft-mediated endocytosis. H292 cells were pretreated with sRPMI containing inhibitor or inhibitor diluent, followed by addition of the cargo conjugate, incubation for 20 min on ice, and incubation for 2 h at 37°C. (C) Adherence-invasion assays. Filipin and nystatin do not inhibit invasion by NTHI. H292 cells were pretreated with inhibitor or inhibitor diluent for 2 h, followed by a 4-h infection conducted according to the adherence-invasion assay protocol. Data are normalized to samples in the absence of inhibitor. Error bars represent standard errors of the means of three independent experiments. A paired Student t test was used to calculate statistical significance. *, P ≤ 0.05; **, P ≤ 0.005.
    Prolong Gold Antifade Reagent, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 50878 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore methyl β cyclodextrin
    Confocal microscopy and adherence-invasion assays with pharmacologic inhibitors. Lipid raft-independent invasion of bronchial epithelial cells by NTHI. (A) Confocal microscopy. At 4 and 24 h postinoculation of H292 cells, NTHI does not colocalize with vesicles positive for the following markers of lipid rafts: caveolin-1 (shown at 24 h), flotillin-1 (shown at 24 h), and cholesterol (shown at 4 h). NTHI was labeled with anti-11P6H antibody conjugated to <t>Alexa</t> <t>Fluor</t> 488 (green). Caveolin-1 was labeled with anti-caveolin-1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). Flotillin-1 was labeled with anti-flotillin-1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). Cholesterol was labeled with filipin (blue), and those samples were also labeled with anti-human secretory component antibody and donkey anti-goat antibody conjugated to Alexa Fluor 568 (red) to provide additional visualization of the plasma membrane. (B) Confocal microscopy. Filipin (FLP) and nystatin (NST) inhibit the internalization of fluorescently conjugated cholera toxin B subunit (white arrows), a known cargo of lipid raft-mediated endocytosis. H292 cells were pretreated with sRPMI containing inhibitor or inhibitor diluent, followed by addition of the cargo conjugate, incubation for 20 min on ice, and incubation for 2 h at 37°C. (C) Adherence-invasion assays. Filipin and nystatin do not inhibit invasion by NTHI. H292 cells were pretreated with inhibitor or inhibitor diluent for 2 h, followed by a 4-h infection conducted according to the adherence-invasion assay protocol. Data are normalized to samples in the absence of inhibitor. Error bars represent standard errors of the means of three independent experiments. A paired Student t test was used to calculate statistical significance. *, P ≤ 0.05; **, P ≤ 0.005.
    Methyl β Cyclodextrin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2169 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore cytochalasin d
    IPNV infection is dependent on actin polymerization dynamics. A. CHSE-214 cells were infected with IPNV at a MOI of 10 and adsorbed for 1 h at 4 °C. The supernatant was removed and MEM medium added at 20 °C. Cells were incubated at 20 °C for 15 min, fixed and stained with rhodamine-conjugated phalloidin. Images were recorded with a C2 Plus Nikon spectral confocal microscope, (scale = 10 μm). ( A ) no infection ( B ) 15 min post infection (white arrow heads indicate stress fibers in A and disorganized actin in B ). ( C ) CHSE-214 cells were preincubated with 1 or 2 µM <t>cytochalasin</t> D (Cyto D) for 1 h and infected with IPNV at a MOI of 1 in the presence of Cyto D. Cells were incubated at 20 °C for 6 h and processed by indirect immunofluorescence (IFI) for IPNV VP2/VP3. For each condition, 250 cells were counted. The number of VP2/VP3-expressing cells is shown as a percentage, normalized to mock cells (****p
    Cytochalasin D, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 9195 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher trypsin edta
    IPNV infection is dependent on actin polymerization dynamics. A. CHSE-214 cells were infected with IPNV at a MOI of 10 and adsorbed for 1 h at 4 °C. The supernatant was removed and MEM medium added at 20 °C. Cells were incubated at 20 °C for 15 min, fixed and stained with rhodamine-conjugated phalloidin. Images were recorded with a C2 Plus Nikon spectral confocal microscope, (scale = 10 μm). ( A ) no infection ( B ) 15 min post infection (white arrow heads indicate stress fibers in A and disorganized actin in B ). ( C ) CHSE-214 cells were preincubated with 1 or 2 µM <t>cytochalasin</t> D (Cyto D) for 1 h and infected with IPNV at a MOI of 1 in the presence of Cyto D. Cells were incubated at 20 °C for 6 h and processed by indirect immunofluorescence (IFI) for IPNV VP2/VP3. For each condition, 250 cells were counted. The number of VP2/VP3-expressing cells is shown as a percentage, normalized to mock cells (****p
    Trypsin Edta, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 49323 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher alexa fluor 488 phalloidin
    Integrin β4 and α6 dimerize during the IR-induced cellular senescence. a–c A549 cells pre-incubated with an inhibitor of integrin β4 (ASC-8) or Src (PP2) were irradiated with 6 Gy IR. SA-β-Gal positivity was observed 3 days after irradiation. Scale bars indicate 10 μm ( a ). Immunoblotting was performed with the indicated antibodies at various time intervals after irradiation. Actin served as the loading control ( b , c ). d A549 cells pre-incubated with 0.5 mM MβCD were irradiated with 6 Gy IR. At 1 h post-irradiation, immunoprecipitates were prepared with anti-α-integrin β4, and then immunoblotted with anti-α-integrin α6. e A549 cells were irradiated with 6 Gy and incubated for 1 h, and lipid rafts were fractionated. Equal volumes of lipid rafts (Rafts; fractions 8–10) and nonlipid rafts (Nonrafts; fractions 3–6) were resolved by SDS-PAGE, and immunoblotting was performed with the indicated antibodies. Actin was used as a marker of nonraft fractions. f A549 cells were irradiated with 6 Gy, incubated for the indicated times, and then labeled with filipin and <t>Alexa</t> <t>Fluor</t> 488 <t>phalloidin.</t> The fluorescence intensities of filipin per area (mm 2 ) were quantified from 15 different cells using the ImageJ program. Scale bars indicate 20 μm . g A549 cells were irradiated with 6 Gy and incubated for the indicated times, and the cholesterol contents in cells were measured using an Amplex Red cholesterol assay kit. The results are expressed with respect to the amount of protein. h , i A549 cells pre-incubated with oleic acid were irradiated with 6 Gy IR. Cell lysates prepared at the indicated time intervals were immunoblotted ( h ), and SA-β-Gal staining ( i ) was performed. Scale bars indicate 10 μm. The values represent the mean ± SD of three independent experiments; *** indicates p
    Alexa Fluor 488 Phalloidin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 12447 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher lysotracker red
    FGF19 inhibits lysosomal stress-induced TFEB nuclear translocation. a HepG2 cells were treated with vehicle (Veh) or 25 µg ml −1 cholesterol for 8 h and stained with <t>lysotracker</t> red and filipin. Images were acquired with a confocal microscope. Scale bar: 30 µm. Images are representative of three independent experiments with similar results. b Nuclear and cytosolic TFEB protein in human hepatocytes and HepG2 cells treated with 25 µg ml −1 cholesterol for 6 h. H3: histone 3. Representative images of at least four independent experiments. c HepG2 cells were transfected with TFEB-GFP expression plasmid. After 24 h, cells were treated with 25 µg ml −1 cholesterol for 8 h or cultured in amino acid free EBSS culture medium for 3 h. Nuclei were stained with DAPI. Left panel, Confocal microscope was used to acquire images. Scale bar: 20 µm. Right panel, average nuclear/total GFP fluorescent intensity of ~80–100 cells. Results were expressed as mean ± SD. d Nuclear and cytosolic TFEB protein. Human hepatocytes were treated with 50 ng ml −1 FGF19 for 30 min followed by 25 µg ml −1 cholesterol treatment for 6 h. Left panel: A representative blot. Right panel: Average nuclear TFEB protein normalized to histone 3 (H3) in four independent preparations of human hepatocytes ( n = 4). Control value was arbitrarily set as 1. e Nuclear and cytosolic TFEB protein in HepG2 cells. Treatment was the same as in d . Left panel: A representative blot. Right panel: Average nuclear TFEB protein normalized to histone 3 (H3) of six independent experiments. Control value was arbitrarily set as 1. f TFEB-FLAG expression plasmids were transfected in HepG2 cells. After overnight culture in serum free medium, cells were treated as in d . Immunostaining was performed with anti-FLAG antibody. Left panel: representative confocal image (Scale bar: 25 µm). Right panel: Average nuclear/total fluorescent intensity of three independent experiments (Total of 250–360 cells were analyzed per condition). All results in d – f were expressed as mean ± SEM. Two-sided Student’s t -test was used for c – f . Source data for b – f are provided as a Source Data file.
    Lysotracker Red, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 4595 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher fm4 64
    Vacuoles contact the void zone. (A) Cortical ER is disassociated from the void zone. Cells expressing mRFP-Snc1-pm and either Hmg1-GFP or Rtn1-GFP were grown in YPDA medium at 37°C. The mRFP-Snc1-pm and ER marker proteins are shown in green and magenta, respectively. Arrowheads indicate the void zone. Scale bar: 5 μm. (B) Vacuoles contact the void zone. Cells expressing GFP-Snc1-pm were grown in YPDA medium at 37°C and stained with <t>FM4-64</t> for 20 min. Arrowheads indicate the void zone. Scale bar: 5 μm. (C) Separation of the vacuolar protein in the void zone contact region. Cells expressing mRFP-Snc1-pm and either Vph1-GFP or GFP-Cot1 and cells expressing GFP-Snc1-pm and mRFP-Zrc1 were grown in YPDA medium at 37°C. Snc1-pm and vacuolar proteins are shown in green and magenta, respectively. Arrowheads indicate the void zone. Scale bar: 5 μm. (D) Time-lapse imaging of the vacuole-void zone contact. cho1 Δ cells expressing GFP-Snc1-pm and Vph1-mRFP were grown in YPDA medium at 37°C. Three examples with two distinctive patterns (a, b) are shown. In the bottom left scheme, black regions in the plasma membrane indicate the void zone. Numbers indicate time in min. Scale bar: 5 μm. (E) The vacuole-void zone contact is stable. Cells observed by time-lapse imaging as in (D) are categorized by the presence or absence of the V-V contact and the void zone behaviour (n > 100 cells with the void zone). Estimation was based on the void zone or the V-V contact lasting more than 30 min during 1 h observation.
    Fm4 64, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 5000 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Detection of genetic changes in post-cell fusion cells and a proposed model of cell fusion in tumor progression (a). Ten single cell clones including 2 pS-puro-scrambled (clones 1-2), 2 pEYFP-N3-neo (clones 3-4), and 6 pS-puro-scrambled-pEYFP-N3-neo double selected clones (clones 5-10) were selected at random and amplified in cell culture. Genomic DNA was extracted from the cells. AmpFlSTR Identifiler PCR Amplification kit was used to examine microsatellite markers. Shown are fragment analysis of the D19S433 alleles. Note the fragment height of allele a (in the red box) was decreased in the fusion cells (6-9), and lost completely in clone #10 (b). A proposed model of K type human endogenous retrovirus in mediating intercellular fusion, evolution of genetic changes, and malignant progression

    Journal: Journal of Carcinogenesis

    Article Title: Human endogenous retroviral K element encodes fusogenic activity in melanoma cells

    doi: 10.4103/1477-3163.109032

    Figure Lengend Snippet: Detection of genetic changes in post-cell fusion cells and a proposed model of cell fusion in tumor progression (a). Ten single cell clones including 2 pS-puro-scrambled (clones 1-2), 2 pEYFP-N3-neo (clones 3-4), and 6 pS-puro-scrambled-pEYFP-N3-neo double selected clones (clones 5-10) were selected at random and amplified in cell culture. Genomic DNA was extracted from the cells. AmpFlSTR Identifiler PCR Amplification kit was used to examine microsatellite markers. Shown are fragment analysis of the D19S433 alleles. Note the fragment height of allele a (in the red box) was decreased in the fusion cells (6-9), and lost completely in clone #10 (b). A proposed model of K type human endogenous retrovirus in mediating intercellular fusion, evolution of genetic changes, and malignant progression

    Article Snippet: Genomic DNA was extracted and examined using AmpFISTR Identifiler PCR Amplification kit (Life Technologies, Carlsbad, CA).

    Techniques: Clone Assay, Amplification, Cell Culture, Polymerase Chain Reaction

    Ezetimibe is protective in a zebrafish model of S . Typhi infection. ( A ) Fish infected with S . Typhi prgH had increased survival compared with fish infected with WT S . Typhi ( P = 0.01). The survival curve was carried out for 5 d; fish were checked once each day. ( B ) Zebrafish were scored 24 h post S . Typhi infection as cleared (no bacteria), localized (bacteria only in the swim bladder), disseminated (bacteria found outside the swim bladder), or dead (fish dead due to bacterial burden). The swim bladders are denoted by red circles; bacteria are denoted by the red arrows. ( C ) Fish infected with the S . Typhi prgH mutant had increased clearance of bacteria at 24 h ( P = 0.03). ( D ) Ezetimibe had no effect on S . Typhi bacterial growth. Bacteria were diluted from an overnight stock and grown with DMSO or 10 µM ezetimibe. The OD 600 was taken every 30 min for 3.5 h. Data points are the mean from two separate experiments. ( E ) Ezetimibe decreased filipin staining in fish. Twenty-four–hour pretreatment with 10 µM ezetimibe reduced filipin (0.05 mg/mL) staining ( P = 0.003) in zebrafish; n = 20 fish from two separate experiments; P value from an unpaired t test. ( F ) Fish pretreated with ezetimibe had increased survival from S . Typhi infection compared with DMSO-pretreated controls ( P = 0.03). ( G ) Ezetimibe treatment increased bacterial clearance in fish. Twenty-four–hour pretreatment with 10 µM ezetimibe increased the percentage of fish that cleared the bacteria 24 h postinfection from 8 to 30% ( P = 0.009). Infection data for each survival curve and clearance comparisons are from three independent experiments with a minimum of n = 60 fish. P values from survival curves are from the Mantel–Cox test; P values for other comparisons are from unpaired t tests.

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

    Article Title: Human genetic variation in VAC14 regulates Salmonella invasion and typhoid fever through modulation of cholesterol

    doi: 10.1073/pnas.1706070114

    Figure Lengend Snippet: Ezetimibe is protective in a zebrafish model of S . Typhi infection. ( A ) Fish infected with S . Typhi prgH had increased survival compared with fish infected with WT S . Typhi ( P = 0.01). The survival curve was carried out for 5 d; fish were checked once each day. ( B ) Zebrafish were scored 24 h post S . Typhi infection as cleared (no bacteria), localized (bacteria only in the swim bladder), disseminated (bacteria found outside the swim bladder), or dead (fish dead due to bacterial burden). The swim bladders are denoted by red circles; bacteria are denoted by the red arrows. ( C ) Fish infected with the S . Typhi prgH mutant had increased clearance of bacteria at 24 h ( P = 0.03). ( D ) Ezetimibe had no effect on S . Typhi bacterial growth. Bacteria were diluted from an overnight stock and grown with DMSO or 10 µM ezetimibe. The OD 600 was taken every 30 min for 3.5 h. Data points are the mean from two separate experiments. ( E ) Ezetimibe decreased filipin staining in fish. Twenty-four–hour pretreatment with 10 µM ezetimibe reduced filipin (0.05 mg/mL) staining ( P = 0.003) in zebrafish; n = 20 fish from two separate experiments; P value from an unpaired t test. ( F ) Fish pretreated with ezetimibe had increased survival from S . Typhi infection compared with DMSO-pretreated controls ( P = 0.03). ( G ) Ezetimibe treatment increased bacterial clearance in fish. Twenty-four–hour pretreatment with 10 µM ezetimibe increased the percentage of fish that cleared the bacteria 24 h postinfection from 8 to 30% ( P = 0.009). Infection data for each survival curve and clearance comparisons are from three independent experiments with a minimum of n = 60 fish. P values from survival curves are from the Mantel–Cox test; P values for other comparisons are from unpaired t tests.

    Article Snippet: Imaging flow cytometry was performed on 1 million cells per sample, which were washed with PBS, fixed with 3.7% PFA for 20 min at RT, and stained with WGA Alexa Fluor 680 (Thermo Fisher) for 10 min, 0.05 mg/mL Filipin (Sigma) for 2 h, or both stains and were run through the ImageStream X Flow Cytometer (Amnis Corp.).

    Techniques: Infection, Fluorescence In Situ Hybridization, Mutagenesis, Staining

    slarp (-) parasites are impaired in liver stage development. (A) HepG2 cells were infected with WT or slarp (-) sporozoites (1×10 4 ) and cultured for 6–72 hours before staining with anti-CSP or anti-HSP70 antibodies and quantification by fluorescence microscopy. Results are expressed as the mean number of parasites from triplicate wells+/−SD. DC, detached infected cells. (B) HepG2 cells were infected with WT or slarp(-) sporozoites and cultured for 24, 48 or 72 hours before staining with anti-HSP70 antibodies (green) and Hoechst 33342 (blue), and examination by confocal fluorescence microscopy. Bar = 10 µm. (C) HepG2 cells were infected with WT or slarp(-) sporozoites and cultured for 24, 48 or 72 hours before staining with anti-HSP70 antibodies and Hoechst 33342. For each population, the number of nuclei in at least 50 parasites was determined by examination under a fluorescence microscope.

    Journal: PLoS Pathogens

    Article Title: A Sporozoite Asparagine-Rich Protein Controls Initiation of Plasmodium Liver Stage Development

    doi: 10.1371/journal.ppat.1000086

    Figure Lengend Snippet: slarp (-) parasites are impaired in liver stage development. (A) HepG2 cells were infected with WT or slarp (-) sporozoites (1×10 4 ) and cultured for 6–72 hours before staining with anti-CSP or anti-HSP70 antibodies and quantification by fluorescence microscopy. Results are expressed as the mean number of parasites from triplicate wells+/−SD. DC, detached infected cells. (B) HepG2 cells were infected with WT or slarp(-) sporozoites and cultured for 24, 48 or 72 hours before staining with anti-HSP70 antibodies (green) and Hoechst 33342 (blue), and examination by confocal fluorescence microscopy. Bar = 10 µm. (C) HepG2 cells were infected with WT or slarp(-) sporozoites and cultured for 24, 48 or 72 hours before staining with anti-HSP70 antibodies and Hoechst 33342. For each population, the number of nuclei in at least 50 parasites was determined by examination under a fluorescence microscope.

    Article Snippet: Hoechst 33342 (Molecular Probes) was used to stain nuclei.

    Techniques: Infection, Cell Culture, Staining, Fluorescence, Microscopy

    SLARP is expressed in P. berghei sporozoites and early/mid LS. (A) RT-PCR analysis of SLARP and TRAP expression in mosquito midgut (MG) and salivary gland (SG) sporozoites. gDNA, genomic DNA; +, with reverse transcription; −, without reverse transcription (B) RT-PCR analysis of SLARP and GAPDH expression in early (24 hours), mid (48 hours) and late (72 hours) LS, merosomes (recovered from the culture supernatants 72 hours post-infection), and mixed blood stages (BS). gDNA, genomic DNA; +, with reverse transcription; −, without reverse transcription. (C) Insertion strategy to generate the SLARP/mCherry parasites. The PbSLARP genomic locus was targeted with an integration plasmid containing a 3′ terminal fragment of the SLARP gene fused in frame to the mCherry coding sequence (M), the 3′ UTR of PbDHFR/TS (3′UTR) and the TgDHFR/TS selectable marker. Upon a single crossover event, the region of homology is duplicated, resulting in a functional, endogenous PbSLARP copy tagged with mCherry, followed by a truncated and non-expressed copy. (D) Detection of the SLARP/mCherry fusion protein (red) was analyzed directly by confocal fluorescence microscopy of sporozoites isolated from mosquito salivary glands (SPZ), or intracellular parasites 2h30 and 24 hours post-infection (p.i.) of HepG2 cells with SLARP/mCherry or WT P. berghei parasites constitutively expressing GFP (green). Nuclei were stained with Hoechst 33342 (blue). Bar = 5 µm. Note that the red fluorescent structures (indicated by asteriks) observed around SLARP/mCherry or WT parasites were also found in non-infected HepG2 cells, and correspond to host autofluorescent material unrelated to mCherry fluorescence (indicated by arrowheads), which was only observed inside SLARP/mCherry (but not WT) parasites.

    Journal: PLoS Pathogens

    Article Title: A Sporozoite Asparagine-Rich Protein Controls Initiation of Plasmodium Liver Stage Development

    doi: 10.1371/journal.ppat.1000086

    Figure Lengend Snippet: SLARP is expressed in P. berghei sporozoites and early/mid LS. (A) RT-PCR analysis of SLARP and TRAP expression in mosquito midgut (MG) and salivary gland (SG) sporozoites. gDNA, genomic DNA; +, with reverse transcription; −, without reverse transcription (B) RT-PCR analysis of SLARP and GAPDH expression in early (24 hours), mid (48 hours) and late (72 hours) LS, merosomes (recovered from the culture supernatants 72 hours post-infection), and mixed blood stages (BS). gDNA, genomic DNA; +, with reverse transcription; −, without reverse transcription. (C) Insertion strategy to generate the SLARP/mCherry parasites. The PbSLARP genomic locus was targeted with an integration plasmid containing a 3′ terminal fragment of the SLARP gene fused in frame to the mCherry coding sequence (M), the 3′ UTR of PbDHFR/TS (3′UTR) and the TgDHFR/TS selectable marker. Upon a single crossover event, the region of homology is duplicated, resulting in a functional, endogenous PbSLARP copy tagged with mCherry, followed by a truncated and non-expressed copy. (D) Detection of the SLARP/mCherry fusion protein (red) was analyzed directly by confocal fluorescence microscopy of sporozoites isolated from mosquito salivary glands (SPZ), or intracellular parasites 2h30 and 24 hours post-infection (p.i.) of HepG2 cells with SLARP/mCherry or WT P. berghei parasites constitutively expressing GFP (green). Nuclei were stained with Hoechst 33342 (blue). Bar = 5 µm. Note that the red fluorescent structures (indicated by asteriks) observed around SLARP/mCherry or WT parasites were also found in non-infected HepG2 cells, and correspond to host autofluorescent material unrelated to mCherry fluorescence (indicated by arrowheads), which was only observed inside SLARP/mCherry (but not WT) parasites.

    Article Snippet: Hoechst 33342 (Molecular Probes) was used to stain nuclei.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Infection, Plasmid Preparation, Sequencing, Marker, Functional Assay, Fluorescence, Microscopy, Isolation, Staining

    Intracellular localization of the Arg-nHAP/DZ1 complex in CNE1-LMP1 cells. Cells were fixed with 4% paraformaldehyde at 4°c for 30 minutes. Nuclei were stained with DAPI (200×). Abbreviations: Arg-nHAP, arginine-modified nanohydroxyapatite particles; DAPI, 4′,6-diamidino-2-phenylindole; DZ1, DNAzyme 1; LF2000, lipofectamine 2000; FITC, fluorescein isothiocyanate.

    Journal: International Journal of Nanomedicine

    Article Title: Delivery system for DNAzymes using arginine-modified hydroxyapatite nanoparticles for therapeutic application in a nasopharyngeal carcinoma model

    doi: 10.2147/IJN.S48321

    Figure Lengend Snippet: Intracellular localization of the Arg-nHAP/DZ1 complex in CNE1-LMP1 cells. Cells were fixed with 4% paraformaldehyde at 4°c for 30 minutes. Nuclei were stained with DAPI (200×). Abbreviations: Arg-nHAP, arginine-modified nanohydroxyapatite particles; DAPI, 4′,6-diamidino-2-phenylindole; DZ1, DNAzyme 1; LF2000, lipofectamine 2000; FITC, fluorescein isothiocyanate.

    Article Snippet: Materials The chemicals, inhibitors, transfection reagents, and cell culture media used in these experiments were sourced as follows: fluorescein isothiocyanate (FITC)-labeled DZ1 (FITC-DZ1) and control DNAzyme (CON) were synthesized by Oligos Etc Inc (Portland, OR, USA); Lipofectamine™ 2000, ProLong® gold antifade reagent with DAPI (4′,6-diamidino-2-phenylindole), and trypsin-EDTA were from Invitrogen Life Technologies (Grand Island, NY, USA); high-performance liquid chromatography grade filipin III ( > 85%), phenylarsine oxide (≥97%), MTS (3-(4,5-di-methylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium), and sodium azide were from Sigma-Aldrich (St Louis, MO, USA); 2-deoxy-D-glucose was from Tokyo Chemical Industry (Tokyo, Japan); Fugene HD was from Roche (Basel, Switzerland); and fetal bovine serum was from Gibco (Grand Island, NY, USA).

    Techniques: Staining, Modification

    Intracellular localization of the Arg-nHAP/DZ1 complex in CNE1-LMP1 cells. Cells were fixed with 4% paraformaldehyde at 4°c for 30 minutes. Nuclei were stained with DAPI (200×). Abbreviations:  Arg-nHAP, arginine-modified nanohydroxyapatite particles; DAPI, 4′,6-diamidino-2-phenylindole; DZ1, DNAzyme 1; LF2000, lipofectamine 2000; FITC, fluorescein isothiocyanate.

    Journal: International Journal of Nanomedicine

    Article Title: Delivery system for DNAzymes using arginine-modified hydroxyapatite nanoparticles for therapeutic application in a nasopharyngeal carcinoma model

    doi: 10.2147/IJN.S48321

    Figure Lengend Snippet: Intracellular localization of the Arg-nHAP/DZ1 complex in CNE1-LMP1 cells. Cells were fixed with 4% paraformaldehyde at 4°c for 30 minutes. Nuclei were stained with DAPI (200×). Abbreviations: Arg-nHAP, arginine-modified nanohydroxyapatite particles; DAPI, 4′,6-diamidino-2-phenylindole; DZ1, DNAzyme 1; LF2000, lipofectamine 2000; FITC, fluorescein isothiocyanate.

    Article Snippet: Materials The chemicals, inhibitors, transfection reagents, and cell culture media used in these experiments were sourced as follows: fluorescein isothiocyanate (FITC)-labeled DZ1 (FITC-DZ1) and control DNAzyme (CON) were synthesized by Oligos Etc Inc (Portland, OR, USA); Lipofectamine™ 2000, ProLong® gold antifade reagent with DAPI (4′,6-diamidino-2-phenylindole), and trypsin-EDTA were from Invitrogen Life Technologies (Grand Island, NY, USA); high-performance liquid chromatography grade filipin III ( > 85%), phenylarsine oxide (≥97%), MTS (3-(4,5-di-methylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium), and sodium azide were from Sigma-Aldrich (St Louis, MO, USA); 2-deoxy-D-glucose was from Tokyo Chemical Industry (Tokyo, Japan); Fugene HD was from Roche (Basel, Switzerland); and fetal bovine serum was from Gibco (Grand Island, NY, USA).

    Techniques: Staining, Modification

    Inhibition of LMP1 protein expression in CNE1-LMP1 cells using DZ1. Cells were transfected with DZ1 or control (40 μg). All values are the mean of three measurements and are shown with error bars. Abbreviations:  Arg-nHAP, arginine-modified nanohydroxyapatite particles; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; LMP1, latent membrane protein 1; DZ1, DNAzyme 1; LF2000, lipofectamine 2000.

    Journal: International Journal of Nanomedicine

    Article Title: Delivery system for DNAzymes using arginine-modified hydroxyapatite nanoparticles for therapeutic application in a nasopharyngeal carcinoma model

    doi: 10.2147/IJN.S48321

    Figure Lengend Snippet: Inhibition of LMP1 protein expression in CNE1-LMP1 cells using DZ1. Cells were transfected with DZ1 or control (40 μg). All values are the mean of three measurements and are shown with error bars. Abbreviations: Arg-nHAP, arginine-modified nanohydroxyapatite particles; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; LMP1, latent membrane protein 1; DZ1, DNAzyme 1; LF2000, lipofectamine 2000.

    Article Snippet: Materials The chemicals, inhibitors, transfection reagents, and cell culture media used in these experiments were sourced as follows: fluorescein isothiocyanate (FITC)-labeled DZ1 (FITC-DZ1) and control DNAzyme (CON) were synthesized by Oligos Etc Inc (Portland, OR, USA); Lipofectamine™ 2000, ProLong® gold antifade reagent with DAPI (4′,6-diamidino-2-phenylindole), and trypsin-EDTA were from Invitrogen Life Technologies (Grand Island, NY, USA); high-performance liquid chromatography grade filipin III ( > 85%), phenylarsine oxide (≥97%), MTS (3-(4,5-di-methylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium), and sodium azide were from Sigma-Aldrich (St Louis, MO, USA); 2-deoxy-D-glucose was from Tokyo Chemical Industry (Tokyo, Japan); Fugene HD was from Roche (Basel, Switzerland); and fetal bovine serum was from Gibco (Grand Island, NY, USA).

    Techniques: Inhibition, Expressing, Transfection, Modification

    Cellular uptake efficiency of the Arg-nHAP/DZ1 complex, cell morphology, and viability. ( A ) Transfected cells were harvested and analyzed using FACS (DZ1, FITC-labeled). FITC fluorescence in the cell population was expressed as the geometric mean of positive events after subtraction of background fluorescence (nontransfected cells). The percentage of FITC-positive cells was determined using the CellQuest software program. All values are the mean of three measurements and are shown with error bars. ( B ) Transfected cells were subjected to fluorescent microscopy, (200×). ( C ) MTS assay was used to assess cellular viability of CNE1-LMP1 cells 24 and 48 hours following treatment with DZ1, Arg-nHAP, Lipofectamine® 2000, Fugene, Arg-nHAP/DZI, Lipofectamine 2000/DZ1 and Fugene/DZ1. The absorbance of each well was read on a microplate reader at 490 nm, and the results indicating the cell viability were plotted as the percentage over controls (MOCK cells). Abbreviations:  FITC, fluorescein isothiocyanate; Arg-nHAP, arginine-modified nanohydroxyapatite particles; DZ1, DNAzyme 1; FACS, fluorescence activated cell sorting; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; LF2000, lipofectamine 2000.

    Journal: International Journal of Nanomedicine

    Article Title: Delivery system for DNAzymes using arginine-modified hydroxyapatite nanoparticles for therapeutic application in a nasopharyngeal carcinoma model

    doi: 10.2147/IJN.S48321

    Figure Lengend Snippet: Cellular uptake efficiency of the Arg-nHAP/DZ1 complex, cell morphology, and viability. ( A ) Transfected cells were harvested and analyzed using FACS (DZ1, FITC-labeled). FITC fluorescence in the cell population was expressed as the geometric mean of positive events after subtraction of background fluorescence (nontransfected cells). The percentage of FITC-positive cells was determined using the CellQuest software program. All values are the mean of three measurements and are shown with error bars. ( B ) Transfected cells were subjected to fluorescent microscopy, (200×). ( C ) MTS assay was used to assess cellular viability of CNE1-LMP1 cells 24 and 48 hours following treatment with DZ1, Arg-nHAP, Lipofectamine® 2000, Fugene, Arg-nHAP/DZI, Lipofectamine 2000/DZ1 and Fugene/DZ1. The absorbance of each well was read on a microplate reader at 490 nm, and the results indicating the cell viability were plotted as the percentage over controls (MOCK cells). Abbreviations: FITC, fluorescein isothiocyanate; Arg-nHAP, arginine-modified nanohydroxyapatite particles; DZ1, DNAzyme 1; FACS, fluorescence activated cell sorting; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; LF2000, lipofectamine 2000.

    Article Snippet: Materials The chemicals, inhibitors, transfection reagents, and cell culture media used in these experiments were sourced as follows: fluorescein isothiocyanate (FITC)-labeled DZ1 (FITC-DZ1) and control DNAzyme (CON) were synthesized by Oligos Etc Inc (Portland, OR, USA); Lipofectamine™ 2000, ProLong® gold antifade reagent with DAPI (4′,6-diamidino-2-phenylindole), and trypsin-EDTA were from Invitrogen Life Technologies (Grand Island, NY, USA); high-performance liquid chromatography grade filipin III ( > 85%), phenylarsine oxide (≥97%), MTS (3-(4,5-di-methylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium), and sodium azide were from Sigma-Aldrich (St Louis, MO, USA); 2-deoxy-D-glucose was from Tokyo Chemical Industry (Tokyo, Japan); Fugene HD was from Roche (Basel, Switzerland); and fetal bovine serum was from Gibco (Grand Island, NY, USA).

    Techniques: Transfection, FACS, Labeling, Fluorescence, Software, Microscopy, MTS Assay, Modification

    Confocal microscopy and adherence-invasion assays with pharmacologic inhibitors. Lipid raft-independent invasion of bronchial epithelial cells by NTHI. (A) Confocal microscopy. At 4 and 24 h postinoculation of H292 cells, NTHI does not colocalize with vesicles positive for the following markers of lipid rafts: caveolin-1 (shown at 24 h), flotillin-1 (shown at 24 h), and cholesterol (shown at 4 h). NTHI was labeled with anti-11P6H antibody conjugated to Alexa Fluor 488 (green). Caveolin-1 was labeled with anti-caveolin-1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). Flotillin-1 was labeled with anti-flotillin-1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). Cholesterol was labeled with filipin (blue), and those samples were also labeled with anti-human secretory component antibody and donkey anti-goat antibody conjugated to Alexa Fluor 568 (red) to provide additional visualization of the plasma membrane. (B) Confocal microscopy. Filipin (FLP) and nystatin (NST) inhibit the internalization of fluorescently conjugated cholera toxin B subunit (white arrows), a known cargo of lipid raft-mediated endocytosis. H292 cells were pretreated with sRPMI containing inhibitor or inhibitor diluent, followed by addition of the cargo conjugate, incubation for 20 min on ice, and incubation for 2 h at 37°C. (C) Adherence-invasion assays. Filipin and nystatin do not inhibit invasion by NTHI. H292 cells were pretreated with inhibitor or inhibitor diluent for 2 h, followed by a 4-h infection conducted according to the adherence-invasion assay protocol. Data are normalized to samples in the absence of inhibitor. Error bars represent standard errors of the means of three independent experiments. A paired Student t test was used to calculate statistical significance. *, P ≤ 0.05; **, P ≤ 0.005.

    Journal: Infection and Immunity

    Article Title: Internalization and Trafficking of Nontypeable Haemophilus influenzae in Human Respiratory Epithelial Cells and Roles of IgA1 Proteases for Optimal Invasion and Persistence

    doi: 10.1128/IAI.00864-13

    Figure Lengend Snippet: Confocal microscopy and adherence-invasion assays with pharmacologic inhibitors. Lipid raft-independent invasion of bronchial epithelial cells by NTHI. (A) Confocal microscopy. At 4 and 24 h postinoculation of H292 cells, NTHI does not colocalize with vesicles positive for the following markers of lipid rafts: caveolin-1 (shown at 24 h), flotillin-1 (shown at 24 h), and cholesterol (shown at 4 h). NTHI was labeled with anti-11P6H antibody conjugated to Alexa Fluor 488 (green). Caveolin-1 was labeled with anti-caveolin-1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). Flotillin-1 was labeled with anti-flotillin-1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). Cholesterol was labeled with filipin (blue), and those samples were also labeled with anti-human secretory component antibody and donkey anti-goat antibody conjugated to Alexa Fluor 568 (red) to provide additional visualization of the plasma membrane. (B) Confocal microscopy. Filipin (FLP) and nystatin (NST) inhibit the internalization of fluorescently conjugated cholera toxin B subunit (white arrows), a known cargo of lipid raft-mediated endocytosis. H292 cells were pretreated with sRPMI containing inhibitor or inhibitor diluent, followed by addition of the cargo conjugate, incubation for 20 min on ice, and incubation for 2 h at 37°C. (C) Adherence-invasion assays. Filipin and nystatin do not inhibit invasion by NTHI. H292 cells were pretreated with inhibitor or inhibitor diluent for 2 h, followed by a 4-h infection conducted according to the adherence-invasion assay protocol. Data are normalized to samples in the absence of inhibitor. Error bars represent standard errors of the means of three independent experiments. A paired Student t test was used to calculate statistical significance. *, P ≤ 0.05; **, P ≤ 0.005.

    Article Snippet: This antibody was purified from rabbit serum using HiTrap protein G HP affinity columns (GE Healthcare) and directly conjugated to Alexa Fluor 488 (Life Technologies).

    Techniques: Confocal Microscopy, Labeling, Incubation, Infection, Invasion Assay

    Confocal microscopy and survival assay with pharmacologic inhibitor of lysosome acidification. NTHI traffics to early endosomes and lysosomes and is killed in lysosomes. IgA1 proteases are required for optimal survival in lysosomes. (A) Confocal microscopy. At 4 and 24 h postinoculation of H292 cells, NTHI is found within vesicles positive for EEA1 (shown at 4 h) and within vesicles positive for LAMP1 (shown at 24 h). NTHI was labeled using anti-11P6H antibody conjugated to Alexa Fluor 488 (green). EEA1 was labeled using anti-EEA1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). LAMP1 was labeled using anti-human LAMP1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). (B) Survival assay in the presence and absence of concanamycin A (CMA). H292 cells were infected for 16 h, treated with gentamicin for 1 h, and treated with concanamycin or diluent for 3 h. Survival data are normalized to samples treated with diluent. Error bars represent standard errors of the means of three independent experiments. A paired Student t test was used to calculate statistical significance. *, P ≤ 0.05; **, P ≤ 0.005.

    Journal: Infection and Immunity

    Article Title: Internalization and Trafficking of Nontypeable Haemophilus influenzae in Human Respiratory Epithelial Cells and Roles of IgA1 Proteases for Optimal Invasion and Persistence

    doi: 10.1128/IAI.00864-13

    Figure Lengend Snippet: Confocal microscopy and survival assay with pharmacologic inhibitor of lysosome acidification. NTHI traffics to early endosomes and lysosomes and is killed in lysosomes. IgA1 proteases are required for optimal survival in lysosomes. (A) Confocal microscopy. At 4 and 24 h postinoculation of H292 cells, NTHI is found within vesicles positive for EEA1 (shown at 4 h) and within vesicles positive for LAMP1 (shown at 24 h). NTHI was labeled using anti-11P6H antibody conjugated to Alexa Fluor 488 (green). EEA1 was labeled using anti-EEA1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). LAMP1 was labeled using anti-human LAMP1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). (B) Survival assay in the presence and absence of concanamycin A (CMA). H292 cells were infected for 16 h, treated with gentamicin for 1 h, and treated with concanamycin or diluent for 3 h. Survival data are normalized to samples treated with diluent. Error bars represent standard errors of the means of three independent experiments. A paired Student t test was used to calculate statistical significance. *, P ≤ 0.05; **, P ≤ 0.005.

    Article Snippet: This antibody was purified from rabbit serum using HiTrap protein G HP affinity columns (GE Healthcare) and directly conjugated to Alexa Fluor 488 (Life Technologies).

    Techniques: Confocal Microscopy, Clonogenic Cell Survival Assay, Labeling, Infection

    IPNV infection is dependent on actin polymerization dynamics. A. CHSE-214 cells were infected with IPNV at a MOI of 10 and adsorbed for 1 h at 4 °C. The supernatant was removed and MEM medium added at 20 °C. Cells were incubated at 20 °C for 15 min, fixed and stained with rhodamine-conjugated phalloidin. Images were recorded with a C2 Plus Nikon spectral confocal microscope, (scale = 10 μm). ( A ) no infection ( B ) 15 min post infection (white arrow heads indicate stress fibers in A and disorganized actin in B ). ( C ) CHSE-214 cells were preincubated with 1 or 2 µM cytochalasin D (Cyto D) for 1 h and infected with IPNV at a MOI of 1 in the presence of Cyto D. Cells were incubated at 20 °C for 6 h and processed by indirect immunofluorescence (IFI) for IPNV VP2/VP3. For each condition, 250 cells were counted. The number of VP2/VP3-expressing cells is shown as a percentage, normalized to mock cells (****p

    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: IPNV infection is dependent on actin polymerization dynamics. A. CHSE-214 cells were infected with IPNV at a MOI of 10 and adsorbed for 1 h at 4 °C. The supernatant was removed and MEM medium added at 20 °C. Cells were incubated at 20 °C for 15 min, fixed and stained with rhodamine-conjugated phalloidin. Images were recorded with a C2 Plus Nikon spectral confocal microscope, (scale = 10 μm). ( A ) no infection ( B ) 15 min post infection (white arrow heads indicate stress fibers in A and disorganized actin in B ). ( C ) CHSE-214 cells were preincubated with 1 or 2 µM cytochalasin D (Cyto D) for 1 h and infected with IPNV at a MOI of 1 in the presence of Cyto D. Cells were incubated at 20 °C for 6 h and processed by indirect immunofluorescence (IFI) for IPNV VP2/VP3. For each condition, 250 cells were counted. The number of VP2/VP3-expressing cells is shown as a percentage, normalized to mock cells (****p

    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, Incubation, Staining, Microscopy, Immunofluorescence, Expressing

    Integrin β4 and α6 dimerize during the IR-induced cellular senescence. a–c A549 cells pre-incubated with an inhibitor of integrin β4 (ASC-8) or Src (PP2) were irradiated with 6 Gy IR. SA-β-Gal positivity was observed 3 days after irradiation. Scale bars indicate 10 μm ( a ). Immunoblotting was performed with the indicated antibodies at various time intervals after irradiation. Actin served as the loading control ( b , c ). d A549 cells pre-incubated with 0.5 mM MβCD were irradiated with 6 Gy IR. At 1 h post-irradiation, immunoprecipitates were prepared with anti-α-integrin β4, and then immunoblotted with anti-α-integrin α6. e A549 cells were irradiated with 6 Gy and incubated for 1 h, and lipid rafts were fractionated. Equal volumes of lipid rafts (Rafts; fractions 8–10) and nonlipid rafts (Nonrafts; fractions 3–6) were resolved by SDS-PAGE, and immunoblotting was performed with the indicated antibodies. Actin was used as a marker of nonraft fractions. f A549 cells were irradiated with 6 Gy, incubated for the indicated times, and then labeled with filipin and Alexa Fluor 488 phalloidin. The fluorescence intensities of filipin per area (mm 2 ) were quantified from 15 different cells using the ImageJ program. Scale bars indicate 20 μm . g A549 cells were irradiated with 6 Gy and incubated for the indicated times, and the cholesterol contents in cells were measured using an Amplex Red cholesterol assay kit. The results are expressed with respect to the amount of protein. h , i A549 cells pre-incubated with oleic acid were irradiated with 6 Gy IR. Cell lysates prepared at the indicated time intervals were immunoblotted ( h ), and SA-β-Gal staining ( i ) was performed. Scale bars indicate 10 μm. The values represent the mean ± SD of three independent experiments; *** indicates p

    Journal: Cell Death and Differentiation

    Article Title: Integrin α6β4-Src-AKT signaling induces cellular senescence by counteracting apoptosis in irradiated tumor cells and tissues

    doi: 10.1038/s41418-018-0114-7

    Figure Lengend Snippet: Integrin β4 and α6 dimerize during the IR-induced cellular senescence. a–c A549 cells pre-incubated with an inhibitor of integrin β4 (ASC-8) or Src (PP2) were irradiated with 6 Gy IR. SA-β-Gal positivity was observed 3 days after irradiation. Scale bars indicate 10 μm ( a ). Immunoblotting was performed with the indicated antibodies at various time intervals after irradiation. Actin served as the loading control ( b , c ). d A549 cells pre-incubated with 0.5 mM MβCD were irradiated with 6 Gy IR. At 1 h post-irradiation, immunoprecipitates were prepared with anti-α-integrin β4, and then immunoblotted with anti-α-integrin α6. e A549 cells were irradiated with 6 Gy and incubated for 1 h, and lipid rafts were fractionated. Equal volumes of lipid rafts (Rafts; fractions 8–10) and nonlipid rafts (Nonrafts; fractions 3–6) were resolved by SDS-PAGE, and immunoblotting was performed with the indicated antibodies. Actin was used as a marker of nonraft fractions. f A549 cells were irradiated with 6 Gy, incubated for the indicated times, and then labeled with filipin and Alexa Fluor 488 phalloidin. The fluorescence intensities of filipin per area (mm 2 ) were quantified from 15 different cells using the ImageJ program. Scale bars indicate 20 μm . g A549 cells were irradiated with 6 Gy and incubated for the indicated times, and the cholesterol contents in cells were measured using an Amplex Red cholesterol assay kit. The results are expressed with respect to the amount of protein. h , i A549 cells pre-incubated with oleic acid were irradiated with 6 Gy IR. Cell lysates prepared at the indicated time intervals were immunoblotted ( h ), and SA-β-Gal staining ( i ) was performed. Scale bars indicate 10 μm. The values represent the mean ± SD of three independent experiments; *** indicates p

    Article Snippet: For staining of cholesterol in cultured cells, cells were fixed with 3% paraformaldehyde for 1 h, washed with cold PBS, incubated with 1.5 mg glycine/mL for 10 min to quench the paraformaldehyde, stained with 0.05 mg/mL filipin (Sigma) for 2 h, and more incubated with Alexa Fluor 488-Phalloidin (1:1000, ThermoFisher Scientific) for 30 min [ ].

    Techniques: Incubation, Irradiation, SDS Page, Marker, Labeling, Fluorescence, Amplex Red Cholesterol Assay, Staining

    FGF19 inhibits lysosomal stress-induced TFEB nuclear translocation. a HepG2 cells were treated with vehicle (Veh) or 25 µg ml −1 cholesterol for 8 h and stained with lysotracker red and filipin. Images were acquired with a confocal microscope. Scale bar: 30 µm. Images are representative of three independent experiments with similar results. b Nuclear and cytosolic TFEB protein in human hepatocytes and HepG2 cells treated with 25 µg ml −1 cholesterol for 6 h. H3: histone 3. Representative images of at least four independent experiments. c HepG2 cells were transfected with TFEB-GFP expression plasmid. After 24 h, cells were treated with 25 µg ml −1 cholesterol for 8 h or cultured in amino acid free EBSS culture medium for 3 h. Nuclei were stained with DAPI. Left panel, Confocal microscope was used to acquire images. Scale bar: 20 µm. Right panel, average nuclear/total GFP fluorescent intensity of ~80–100 cells. Results were expressed as mean ± SD. d Nuclear and cytosolic TFEB protein. Human hepatocytes were treated with 50 ng ml −1 FGF19 for 30 min followed by 25 µg ml −1 cholesterol treatment for 6 h. Left panel: A representative blot. Right panel: Average nuclear TFEB protein normalized to histone 3 (H3) in four independent preparations of human hepatocytes ( n = 4). Control value was arbitrarily set as 1. e Nuclear and cytosolic TFEB protein in HepG2 cells. Treatment was the same as in d . Left panel: A representative blot. Right panel: Average nuclear TFEB protein normalized to histone 3 (H3) of six independent experiments. Control value was arbitrarily set as 1. f TFEB-FLAG expression plasmids were transfected in HepG2 cells. After overnight culture in serum free medium, cells were treated as in d . Immunostaining was performed with anti-FLAG antibody. Left panel: representative confocal image (Scale bar: 25 µm). Right panel: Average nuclear/total fluorescent intensity of three independent experiments (Total of 250–360 cells were analyzed per condition). All results in d – f were expressed as mean ± SEM. Two-sided Student’s t -test was used for c – f . Source data for b – f are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: An FGF15/19-TFEB regulatory loop controls hepatic cholesterol and bile acid homeostasis

    doi: 10.1038/s41467-020-17363-6

    Figure Lengend Snippet: FGF19 inhibits lysosomal stress-induced TFEB nuclear translocation. a HepG2 cells were treated with vehicle (Veh) or 25 µg ml −1 cholesterol for 8 h and stained with lysotracker red and filipin. Images were acquired with a confocal microscope. Scale bar: 30 µm. Images are representative of three independent experiments with similar results. b Nuclear and cytosolic TFEB protein in human hepatocytes and HepG2 cells treated with 25 µg ml −1 cholesterol for 6 h. H3: histone 3. Representative images of at least four independent experiments. c HepG2 cells were transfected with TFEB-GFP expression plasmid. After 24 h, cells were treated with 25 µg ml −1 cholesterol for 8 h or cultured in amino acid free EBSS culture medium for 3 h. Nuclei were stained with DAPI. Left panel, Confocal microscope was used to acquire images. Scale bar: 20 µm. Right panel, average nuclear/total GFP fluorescent intensity of ~80–100 cells. Results were expressed as mean ± SD. d Nuclear and cytosolic TFEB protein. Human hepatocytes were treated with 50 ng ml −1 FGF19 for 30 min followed by 25 µg ml −1 cholesterol treatment for 6 h. Left panel: A representative blot. Right panel: Average nuclear TFEB protein normalized to histone 3 (H3) in four independent preparations of human hepatocytes ( n = 4). Control value was arbitrarily set as 1. e Nuclear and cytosolic TFEB protein in HepG2 cells. Treatment was the same as in d . Left panel: A representative blot. Right panel: Average nuclear TFEB protein normalized to histone 3 (H3) of six independent experiments. Control value was arbitrarily set as 1. f TFEB-FLAG expression plasmids were transfected in HepG2 cells. After overnight culture in serum free medium, cells were treated as in d . Immunostaining was performed with anti-FLAG antibody. Left panel: representative confocal image (Scale bar: 25 µm). Right panel: Average nuclear/total fluorescent intensity of three independent experiments (Total of 250–360 cells were analyzed per condition). All results in d – f were expressed as mean ± SEM. Two-sided Student’s t -test was used for c – f . Source data for b – f are provided as a Source Data file.

    Article Snippet: Lysotracker red was purchased from Thermo-Fisher Scientific (Waltham, MA).

    Techniques: Translocation Assay, Staining, Microscopy, Transfection, Expressing, Plasmid Preparation, Cell Culture, Immunostaining

    Vacuoles contact the void zone. (A) Cortical ER is disassociated from the void zone. Cells expressing mRFP-Snc1-pm and either Hmg1-GFP or Rtn1-GFP were grown in YPDA medium at 37°C. The mRFP-Snc1-pm and ER marker proteins are shown in green and magenta, respectively. Arrowheads indicate the void zone. Scale bar: 5 μm. (B) Vacuoles contact the void zone. Cells expressing GFP-Snc1-pm were grown in YPDA medium at 37°C and stained with FM4-64 for 20 min. Arrowheads indicate the void zone. Scale bar: 5 μm. (C) Separation of the vacuolar protein in the void zone contact region. Cells expressing mRFP-Snc1-pm and either Vph1-GFP or GFP-Cot1 and cells expressing GFP-Snc1-pm and mRFP-Zrc1 were grown in YPDA medium at 37°C. Snc1-pm and vacuolar proteins are shown in green and magenta, respectively. Arrowheads indicate the void zone. Scale bar: 5 μm. (D) Time-lapse imaging of the vacuole-void zone contact. cho1 Δ cells expressing GFP-Snc1-pm and Vph1-mRFP were grown in YPDA medium at 37°C. Three examples with two distinctive patterns (a, b) are shown. In the bottom left scheme, black regions in the plasma membrane indicate the void zone. Numbers indicate time in min. Scale bar: 5 μm. (E) The vacuole-void zone contact is stable. Cells observed by time-lapse imaging as in (D) are categorized by the presence or absence of the V-V contact and the void zone behaviour (n > 100 cells with the void zone). Estimation was based on the void zone or the V-V contact lasting more than 30 min during 1 h observation.

    Journal: bioRxiv

    Article Title: Phosphatidylserine prevents the generation of a protein-free giant plasma membrane domain in yeast

    doi: 10.1101/2020.08.10.245530

    Figure Lengend Snippet: Vacuoles contact the void zone. (A) Cortical ER is disassociated from the void zone. Cells expressing mRFP-Snc1-pm and either Hmg1-GFP or Rtn1-GFP were grown in YPDA medium at 37°C. The mRFP-Snc1-pm and ER marker proteins are shown in green and magenta, respectively. Arrowheads indicate the void zone. Scale bar: 5 μm. (B) Vacuoles contact the void zone. Cells expressing GFP-Snc1-pm were grown in YPDA medium at 37°C and stained with FM4-64 for 20 min. Arrowheads indicate the void zone. Scale bar: 5 μm. (C) Separation of the vacuolar protein in the void zone contact region. Cells expressing mRFP-Snc1-pm and either Vph1-GFP or GFP-Cot1 and cells expressing GFP-Snc1-pm and mRFP-Zrc1 were grown in YPDA medium at 37°C. Snc1-pm and vacuolar proteins are shown in green and magenta, respectively. Arrowheads indicate the void zone. Scale bar: 5 μm. (D) Time-lapse imaging of the vacuole-void zone contact. cho1 Δ cells expressing GFP-Snc1-pm and Vph1-mRFP were grown in YPDA medium at 37°C. Three examples with two distinctive patterns (a, b) are shown. In the bottom left scheme, black regions in the plasma membrane indicate the void zone. Numbers indicate time in min. Scale bar: 5 μm. (E) The vacuole-void zone contact is stable. Cells observed by time-lapse imaging as in (D) are categorized by the presence or absence of the V-V contact and the void zone behaviour (n > 100 cells with the void zone). Estimation was based on the void zone or the V-V contact lasting more than 30 min during 1 h observation.

    Article Snippet: For staining of the plasma membrane by FM4-64 (Thermo Fisher Scientific, MA, USA), cell suspensions were mixed with an equal volume of 100 μM FM4-64 on a glass slide and observed immediately.

    Techniques: Expressing, Marker, Staining, Imaging