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  • 99
    Carl Zeiss confocal microscope
    Live-cell images demonstrating intercellular transport of PRRSV GFP-tagged nsp2 through nanotubes. (A) GFP-PRRSV-infected cells maintained in culture medium containing PRRSV-neutralizing antibody. (B) GFP-PRRSV-infected cells maintained in regular cultural medium (no PRRSV-neutralizing antibody). (C) HEK-293T cells were <t>transfected</t> with a PRRSV full-length cDNA infectious clone, pCMV-SD95-21-GFP. At 24 h postinfection (A and B) or transfection (C), cells were analyzed using a live-cell image system of a confocal microscope (LSM 880; <t>Zeiss).</t> In both infected MARC-145 cells and transfected HEK-293T cells, the dot-like GFP-nsp2 proteins were visualized as moving through an intercellular nanotube connection (arrows) into the cytoplasm of a neighboring cell. Insets show a zoomed area of interest that contains the GFP-nsp2 proteins. The specific nanotube shown between MARC-145 cells is about 20 μm in length, while the specific nanotube shown between HEK-293T cells is about 12 μm in length. Scale bar, 10 μm.
    Confocal Microscope, supplied by Carl Zeiss, used in various techniques. Bioz Stars score: 99/100, based on 49321 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Carl Zeiss confocal laser scanning microscope
    Combined immunolocalization of Er-PDF-I and Er-PDF-I in the brain of E. rowelli . <t>Confocal</t> <t>laser</t> <t>scanning</t> micrographs of vibratome sections. Dorsal is up in all images and anterior is left in (B,G) . Er-PDF-I-ir (magenta), Er-PDF-II-ir (green) and DNA (gray). (A,B) Er-PDF-I and Er-PDF-II are co-localized in neuropil and somata, albeit at different intensities. (C) Somata (arrowheads) and processes (arrows) of dorsal cell group exhibit equal intensity levels of Er-PDF-I-ir and Er-PDF-II-ir. (D) In contrast, somata and processes of ventral cell group occur in three variants: (i) Er-PDF-I-ir and Er-PDF-II-ir at equal levels (open arrowheads), (ii) Er-PDF-I-ir at higher level (arrows), and (iii) Er-PDF-II-ir at higher level (filled arrowheads). (E–H) Differences in expression levels of Er-PDF-I-ir and Er-PDF-I-ir are also seen in optic neuropil (E) , inner lobe (black asterisk) as opposed to remaining lobes of the mushroom bodies (white asterisks) (F) , somata of connecting cords (G) , and somata and neuropil of nerve cords (H) . an, anterior neuropil; ad, anterior division of central body; at, antennal tract; cb, central body; cc, connecting cord; cn, central neuropil; dl, dorsal perikaryal layer; ey, eye; mb, mushroom body; ol, olfactory lobe; on, optic neuropil; ot, optic tract; pd, posterior division of the central body; vl, ventral perikaryal layer; vn, neuropil of ventral nerve cord; vp, perikaryal layer of ventral nerve cord. Scale bars: 50 μm (A,B) and 20 μm (C–H) .
    Confocal Laser Scanning Microscope, supplied by Carl Zeiss, used in various techniques. Bioz Stars score: 95/100, based on 39975 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Olympus confocal microscope
    Combined immunolocalization of Er-PDF-I and Er-PDF-I in the brain of E. rowelli . <t>Confocal</t> <t>laser</t> <t>scanning</t> micrographs of vibratome sections. Dorsal is up in all images and anterior is left in (B,G) . Er-PDF-I-ir (magenta), Er-PDF-II-ir (green) and DNA (gray). (A,B) Er-PDF-I and Er-PDF-II are co-localized in neuropil and somata, albeit at different intensities. (C) Somata (arrowheads) and processes (arrows) of dorsal cell group exhibit equal intensity levels of Er-PDF-I-ir and Er-PDF-II-ir. (D) In contrast, somata and processes of ventral cell group occur in three variants: (i) Er-PDF-I-ir and Er-PDF-II-ir at equal levels (open arrowheads), (ii) Er-PDF-I-ir at higher level (arrows), and (iii) Er-PDF-II-ir at higher level (filled arrowheads). (E–H) Differences in expression levels of Er-PDF-I-ir and Er-PDF-I-ir are also seen in optic neuropil (E) , inner lobe (black asterisk) as opposed to remaining lobes of the mushroom bodies (white asterisks) (F) , somata of connecting cords (G) , and somata and neuropil of nerve cords (H) . an, anterior neuropil; ad, anterior division of central body; at, antennal tract; cb, central body; cc, connecting cord; cn, central neuropil; dl, dorsal perikaryal layer; ey, eye; mb, mushroom body; ol, olfactory lobe; on, optic neuropil; ot, optic tract; pd, posterior division of the central body; vl, ventral perikaryal layer; vn, neuropil of ventral nerve cord; vp, perikaryal layer of ventral nerve cord. Scale bars: 50 μm (A,B) and 20 μm (C–H) .
    Confocal Microscope, supplied by Olympus, used in various techniques. Bioz Stars score: 96/100, based on 21812 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Carl Zeiss lsm 510 confocal microscope
    Cathepsin X enables podosome formation. Immature DCs on Day 5 of differentiation were centrifuged with cytospin (Cytofuge) for 6 min at 1000 rpm onto glass cover slides. Actin was labeled with phalloidin-tetramethylrhodamine B isothiocyanate conjugate (500 ng/ml) for 30 min at room temperature. Podosome formation, present in control, immature DCs (A), is prevented by inhibition of cathepsin X during DC differentiation (B). Adhesion of maturing DCs coincides with β 2 -integrin activation and colocalization with actin (C). Immature and mature DCs were labeled by centrifugation with cytospin (Cytofuge), whereas maturing, adherent DCs were labeled by seeding immature DCs onto glass coverslips in 24-well plates in the presence of 20 ng/ml LPS and allowing adherence for 20 h. The active form of β 2 integrin was labeled with mAb 24 (green fluorescence) and colocalized with actin (red fluorescence) in adherent, mature DCs. Meanwhile, formation of typical dendrites in mature DCs (D) was not inhibited by cathepsin X inhibition (E). Original scale bars represent 20 μm. Fluorescence microscopy was performed using a Carl Zeiss <t>LSM</t> 510 confocal microscope. Images were analyzed using Carl Zeiss LSM image software 3.0.
    Lsm 510 Confocal Microscope, supplied by Carl Zeiss, used in various techniques. Bioz Stars score: 96/100, based on 25755 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Olympus confocal laser scanning microscope
    Localization of Mel-AF on A375 cells was analyzed using a <t>confocal</t> <t>laser</t> <t>scanning</t> <t>microscope.</t> The cells were treated with FITC-conjugated Mel-AF for 2 h. ( A ) Confocal images show the localization of Mel-AF traced with FITC (green). The membrane compartment was indicated by staining with a specific antibody to CD46, a membrane cofactor protein followed by Alexa Fluor 568 rabbit anti-mouse IgG (red). The cellular nuclei were counterstained by DAPI (blue). ( B ) Orthogonal imaging analysis was performed to confirm the localization of Mel-AF on the cell membrane (yellow). Scale bar, 10 μm.
    Confocal Laser Scanning Microscope, supplied by Olympus, used in various techniques. Bioz Stars score: 96/100, based on 18562 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Carl Zeiss lsm 710 confocal microscope
    Effects of recent transfection with anti-EGFP siRNA on SFV at 4 h post infection. CHO cells were transfected with siRNA targeting EGFP mRNA using PF6 (B, C, D) or LF2000 (E, F, G) reagents. Transfected cells were infected with SFV(ZsGreen) 4 at an MOI of 0.1 at 2 h (B, E), 4 h (C, F) or 8 h (D, G) post transfection. All cells were fixed at 4 h post infection. The localization of nsP1 (red) of SFV was revealed using rabbit polyclonal antiserum as the primary detection reagent and Alexa 568 conjugated anti-rabbit antibody as the secondary antibody; ZsGreen was detected by its green fluorescence; the co-localization of these signals is shown as yellow. Nuclei were counter-stained with DAPI (blue). Images were collected using a <t>LSM</t> 710 confocal microscope (Zeiss); scale bar represents 10 µm. A single optical slice from a characteristic infected cell is shown in each panel, and the nuclei of several non-infected cells are also visible. Panel A shows a representative cell from the control experiment (no transfection, cell fixed 4 h after infection with SFV(ZsGreen) 4).
    Lsm 710 Confocal Microscope, supplied by Carl Zeiss, used in various techniques. Bioz Stars score: 96/100, based on 17769 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Olympus fv1000 confocal microscope
    The N-terminal 63 amino acids of UL31 are sufficient to recruit EGFP to sites of laser microirradiation. Images of a live HeLa cell transfected with EGFP-N63 immediately before (Pre), immediately after (Post), and 30 seconds were acquired after the area indicated by the arrowhead was irradiated using an Olympus <t>FV1000</t> laser-scanning confocal microscope. Images shown are representative of n = 12 cells analyzed in two independent experiments. Error bars are standard error of the mean (n = 12).
    Fv1000 Confocal Microscope, supplied by Olympus, used in various techniques. Bioz Stars score: 95/100, based on 12152 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Carl Zeiss confocal microscopy
    The N-terminal 63 amino acids of UL31 are sufficient to recruit EGFP to sites of laser microirradiation. Images of a live HeLa cell transfected with EGFP-N63 immediately before (Pre), immediately after (Post), and 30 seconds were acquired after the area indicated by the arrowhead was irradiated using an Olympus <t>FV1000</t> laser-scanning confocal microscope. Images shown are representative of n = 12 cells analyzed in two independent experiments. Error bars are standard error of the mean (n = 12).
    Confocal Microscopy, supplied by Carl Zeiss, used in various techniques. Bioz Stars score: 94/100, based on 21695 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Carl Zeiss lsm 700 confocal microscope
    ( a ) Serum-deprived cells present a quiescent phenotype to manage oxidative stress. (i) One hundred fifty thousand (1.5 × 10 5 ) PC3 prostate cancer cells were plated with or without serum (24 h) prior to treatment with 250 μM H 2 0 2 for 4 h. Phase contrast microscopy (20×) was captured cell morphology. (ii) One million (1 × 10 6 ) serum-containing and serum-deprived PC3 prostate cancer cells were pre-treated with 5 mM n-acetyl-cysteine (NAC) for 1 h prior to treatment with 250 μM H 2 O 2 . Quiescence was determined via Western blot analysis for phosphorylated-retinoblastoma (pRB) or p27 Kip1 protein expression. β-actin served as a loading control. Pictures of gels/blots were cropped to focus on target protein expression. Full length gels/blots are included in supplementary figure 1 . (iii) One hundred fifty thousand (1.5 × 10 5 ) cells were treated as mentioned above and fixed in 4% paraformaldehyde prior to incubation with rabbit anti-p27 Kip1 and mouse anti-PMCA antibodies, followed by Cy3-conjugated anti-rabbit and Alexa Fluor-488 anti-mouse antibodies. Imaging of cells (63×) was performed on a Zeiss LSM700 Confocal Microscope. Scale bar = 50 μm. ( b ) Serum-deprived cells present a quiescent phenotype to manage oxidative stress. (i) One million (1.0 × 10 6 ) DU145 prostate cancer cells were plated with or without serum (24 h) prior to treatment with 250 μM H 2 0 2 for 4 h. Phase contrast microscopy (20×) was utilized to captured cell morphology. (ii) One million (1 × 10 6 ) serum-containing and serum-deprived DU145 prostate cancer cells were pre-treated with 5 mM n-acetyl-cysteine (NAC) for 1 h prior to treatment with 250 μM H 2 O 2 . Quiescence was determined via Western blot analysis for phosphorylated-retinoblastoma (pRB) or p27 Kip1 protein expression. β-actin served as a loading control. Pictures of gels/blots were cropped to focus on target protein expression. Full length gels/blots are included in supplementary figure 2 . (iii) One hundred fifty thousand (1.5 × 10 5 ) DU145 prostate cancer cells were treated as mentioned above and fixed in 4% paraformaldehyde prior to incubation with rabbit anti-p27 Kip1 and mouse anti-PMCA antibodies, followed by Cy3-conjugated anti-rabbit and Alexa Fluor-488 anti-mouse antibodies. Imaging of cells (63×) was performed on a Zeiss <t>LSM-700</t> Confocal Microscope. Scale bar = 50 μm.
    Lsm 700 Confocal Microscope, supplied by Carl Zeiss, used in various techniques. Bioz Stars score: 95/100, based on 12032 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Carl Zeiss lsm 780 confocal microscope
    Immunolocalization of both ataxin‐2 B and C in the fat body cells of third instar larvae. Immunolabeling using α‐dAtx2 B + C and anti‐rabbit IgG conjugated to Alexa 488 (green). Actin filaments were stained with phalloidin conjugated to Alexa 594 (red) and nuclei with 4′,6‐diamidino‐2‐phenylindole, fluorescent stain that binds strongly to A‐T rich regions in DNA (blue). The images were captured using the Zeiss <t>LSM</t> 780 confocal microscope.
    Lsm 780 Confocal Microscope, supplied by Carl Zeiss, used in various techniques. Bioz Stars score: 94/100, based on 8523 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Olympus fluoview fv1000 confocal microscope
    Anti-HIV-1 activity, multimerization, and subcellular localization of SWAP1 and SWAP2. ( a ) Effects of chimeric TRIMCyp proteins on HIV-1 infection. CRFK cells stably expressing pLPCX or indicated TRIMCyp proteins were either mock-infected or infected with serial dilutions of VSV-G pseudotyped HIV-1-GFP virus overnight. Forty-eight hours post-infection, infected cells were sorted by FACS. The results are representative of 4 independent experiments. ( b ) Multimerization of SWAP1 and SWAP2. Lysates from CRFK cells stably expressing TRIMCyp proteins were cross-linked using increasing concentrations of glutaraldehyde (0, 0.25, 0.5, 1, or 2 mM) for 3 minutes at 37°C. The reactions were terminated with the addition of 1 M Tris-HCl (pH 8.0), and cross-linked products were subjected to SDS-PAGE and visualized by Western blot using a mouse anti-HA antibody. Asterisk (*) indicates high-order multimers. Results are representative of 2 independent experiments. ( c ) Subcellular localization of SWAP1 and SWAP2. CRFK cells stably expressing TRIMCyp proteins were fixed and immunostained with a mouse anti-HA antibody (green). All images were obtained using an Olympus <t>FluoView</t> <t>FV1000</t> confocal microscope. Nuclei were stained with DAPI (blue). The scale bar indicates 50 μm.
    Fluoview Fv1000 Confocal Microscope, supplied by Olympus, used in various techniques. Bioz Stars score: 95/100, based on 7845 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Leica Microsystems confocal microscope
    Anti-HIV-1 activity, multimerization, and subcellular localization of SWAP1 and SWAP2. ( a ) Effects of chimeric TRIMCyp proteins on HIV-1 infection. CRFK cells stably expressing pLPCX or indicated TRIMCyp proteins were either mock-infected or infected with serial dilutions of VSV-G pseudotyped HIV-1-GFP virus overnight. Forty-eight hours post-infection, infected cells were sorted by FACS. The results are representative of 4 independent experiments. ( b ) Multimerization of SWAP1 and SWAP2. Lysates from CRFK cells stably expressing TRIMCyp proteins were cross-linked using increasing concentrations of glutaraldehyde (0, 0.25, 0.5, 1, or 2 mM) for 3 minutes at 37°C. The reactions were terminated with the addition of 1 M Tris-HCl (pH 8.0), and cross-linked products were subjected to SDS-PAGE and visualized by Western blot using a mouse anti-HA antibody. Asterisk (*) indicates high-order multimers. Results are representative of 2 independent experiments. ( c ) Subcellular localization of SWAP1 and SWAP2. CRFK cells stably expressing TRIMCyp proteins were fixed and immunostained with a mouse anti-HA antibody (green). All images were obtained using an Olympus <t>FluoView</t> <t>FV1000</t> confocal microscope. Nuclei were stained with DAPI (blue). The scale bar indicates 50 μm.
    Confocal Microscope, supplied by Leica Microsystems, used in various techniques. Bioz Stars score: 94/100, based on 5897 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Olympus laser scanning confocal microscopy
    Anti-HIV-1 activity, multimerization, and subcellular localization of SWAP1 and SWAP2. ( a ) Effects of chimeric TRIMCyp proteins on HIV-1 infection. CRFK cells stably expressing pLPCX or indicated TRIMCyp proteins were either mock-infected or infected with serial dilutions of VSV-G pseudotyped HIV-1-GFP virus overnight. Forty-eight hours post-infection, infected cells were sorted by FACS. The results are representative of 4 independent experiments. ( b ) Multimerization of SWAP1 and SWAP2. Lysates from CRFK cells stably expressing TRIMCyp proteins were cross-linked using increasing concentrations of glutaraldehyde (0, 0.25, 0.5, 1, or 2 mM) for 3 minutes at 37°C. The reactions were terminated with the addition of 1 M Tris-HCl (pH 8.0), and cross-linked products were subjected to SDS-PAGE and visualized by Western blot using a mouse anti-HA antibody. Asterisk (*) indicates high-order multimers. Results are representative of 2 independent experiments. ( c ) Subcellular localization of SWAP1 and SWAP2. CRFK cells stably expressing TRIMCyp proteins were fixed and immunostained with a mouse anti-HA antibody (green). All images were obtained using an Olympus <t>FluoView</t> <t>FV1000</t> confocal microscope. Nuclei were stained with DAPI (blue). The scale bar indicates 50 μm.
    Laser Scanning Confocal Microscopy, supplied by Olympus, used in various techniques. Bioz Stars score: 92/100, based on 2682 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Carl Zeiss lsm 510 laser scanning confocal microscope
    Optical sections of an isolated salt gland of  A .  officinalis . Sections (A-E) were taken in the fluorescent mode to show the side views of a salt gland. Details within cells could be observed due to the green autofluorescent nature of the salt gland. Note the presence of strongly autofluorescent structures (arrows) in the middle or periphery of some secretory cells. (F) Corresponding bright-field image of (C) taken together with overlapping image recorded simultaneously in the transmission and fluorescent modes. Optical sections were taken with Zeiss LSM 510. Excitation and emission wavelengths were 488 nm (10%) and  > 530 nm, respectively. Bars = 5 μm.
    Lsm 510 Laser Scanning Confocal Microscope, supplied by Carl Zeiss, used in various techniques. Bioz Stars score: 92/100, based on 6006 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Leica Microsystems confocal laser scanning microscope
    Optical sections of an isolated salt gland of  A .  officinalis . Sections (A-E) were taken in the fluorescent mode to show the side views of a salt gland. Details within cells could be observed due to the green autofluorescent nature of the salt gland. Note the presence of strongly autofluorescent structures (arrows) in the middle or periphery of some secretory cells. (F) Corresponding bright-field image of (C) taken together with overlapping image recorded simultaneously in the transmission and fluorescent modes. Optical sections were taken with Zeiss LSM 510. Excitation and emission wavelengths were 488 nm (10%) and  > 530 nm, respectively. Bars = 5 μm.
    Confocal Laser Scanning Microscope, supplied by Leica Microsystems, used in various techniques. Bioz Stars score: 99/100, based on 4985 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Olympus fluoview 1000 confocal microscope
    Double staining for FASN and NS3. HEK293T/17 cells were pre-treated with 10 μM orlistat or not treated before mock infection or infection with DENV 2. At ( a ) 24 or ( b ) 36 h.p.i cells were fixed and stained to show the FASN ( green ), DENV 2 NS3 protein ( red ) and nuclei ( blue ). Cells were examined under an Olympus <t>FluoView</t> 1000 confocal microscope with 60× magnification. Representative, non-contrast adjusted merged images are shown
    Fluoview 1000 Confocal Microscope, supplied by Olympus, used in various techniques. Bioz Stars score: 94/100, based on 5055 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Live-cell images demonstrating intercellular transport of PRRSV GFP-tagged nsp2 through nanotubes. (A) GFP-PRRSV-infected cells maintained in culture medium containing PRRSV-neutralizing antibody. (B) GFP-PRRSV-infected cells maintained in regular cultural medium (no PRRSV-neutralizing antibody). (C) HEK-293T cells were transfected with a PRRSV full-length cDNA infectious clone, pCMV-SD95-21-GFP. At 24 h postinfection (A and B) or transfection (C), cells were analyzed using a live-cell image system of a confocal microscope (LSM 880; Zeiss). In both infected MARC-145 cells and transfected HEK-293T cells, the dot-like GFP-nsp2 proteins were visualized as moving through an intercellular nanotube connection (arrows) into the cytoplasm of a neighboring cell. Insets show a zoomed area of interest that contains the GFP-nsp2 proteins. The specific nanotube shown between MARC-145 cells is about 20 μm in length, while the specific nanotube shown between HEK-293T cells is about 12 μm in length. Scale bar, 10 μm.

    Journal: Journal of Virology

    Article Title: Porcine Reproductive and Respiratory Syndrome Virus Utilizes Nanotubes for Intercellular Spread

    doi: 10.1128/JVI.00036-16

    Figure Lengend Snippet: Live-cell images demonstrating intercellular transport of PRRSV GFP-tagged nsp2 through nanotubes. (A) GFP-PRRSV-infected cells maintained in culture medium containing PRRSV-neutralizing antibody. (B) GFP-PRRSV-infected cells maintained in regular cultural medium (no PRRSV-neutralizing antibody). (C) HEK-293T cells were transfected with a PRRSV full-length cDNA infectious clone, pCMV-SD95-21-GFP. At 24 h postinfection (A and B) or transfection (C), cells were analyzed using a live-cell image system of a confocal microscope (LSM 880; Zeiss). In both infected MARC-145 cells and transfected HEK-293T cells, the dot-like GFP-nsp2 proteins were visualized as moving through an intercellular nanotube connection (arrows) into the cytoplasm of a neighboring cell. Insets show a zoomed area of interest that contains the GFP-nsp2 proteins. The specific nanotube shown between MARC-145 cells is about 20 μm in length, while the specific nanotube shown between HEK-293T cells is about 12 μm in length. Scale bar, 10 μm.

    Article Snippet: For live-cell movies, infected MARC-145 cells or transfected HEK-293T cells were set into an open cultivation system of the Zeiss confocal microscope and maintained in warm DMEM buffered with HEPES.

    Techniques: Infection, Transfection, Microscopy

    Intercellular transport of the GFP-nsp2 protein in cocultured MARC-145 and HEK-293T cells. (A) GFP-PRRSV-infected MARC-145 cells were fixed and stained with anti-nsp2 MAb 140-68 and anti-SV40 T antigen pAb sc-20800. (B) GFP-PRRSV-infected HEK-293T cells were fixed and stained with anti-nsp2 MAb 140-68 and anti-SV40 T antigen pAb sc-20800. (C) GFP-PRRSV-infected MARC-145 cells were trypsinized at 12 hpi and mixed with naive HEK-293T cells. At 36 h postcultivation, cells were fixed and stained with anti-nsp2 MAb 140-68 and anti-SV40 T antigen pAb sc-20800. The nsp2 was labeled with green fluorescence, and SV40 large T antigen was labeled with red fluorescence. Pictures were taken by a confocal microscope (LSM 880; Zeiss). Scale bar, 10 μm.

    Journal: Journal of Virology

    Article Title: Porcine Reproductive and Respiratory Syndrome Virus Utilizes Nanotubes for Intercellular Spread

    doi: 10.1128/JVI.00036-16

    Figure Lengend Snippet: Intercellular transport of the GFP-nsp2 protein in cocultured MARC-145 and HEK-293T cells. (A) GFP-PRRSV-infected MARC-145 cells were fixed and stained with anti-nsp2 MAb 140-68 and anti-SV40 T antigen pAb sc-20800. (B) GFP-PRRSV-infected HEK-293T cells were fixed and stained with anti-nsp2 MAb 140-68 and anti-SV40 T antigen pAb sc-20800. (C) GFP-PRRSV-infected MARC-145 cells were trypsinized at 12 hpi and mixed with naive HEK-293T cells. At 36 h postcultivation, cells were fixed and stained with anti-nsp2 MAb 140-68 and anti-SV40 T antigen pAb sc-20800. The nsp2 was labeled with green fluorescence, and SV40 large T antigen was labeled with red fluorescence. Pictures were taken by a confocal microscope (LSM 880; Zeiss). Scale bar, 10 μm.

    Article Snippet: For live-cell movies, infected MARC-145 cells or transfected HEK-293T cells were set into an open cultivation system of the Zeiss confocal microscope and maintained in warm DMEM buffered with HEPES.

    Techniques: Infection, Staining, Labeling, Fluorescence, Microscopy

    Combined immunolocalization of Er-PDF-I and Er-PDF-I in the brain of E. rowelli . Confocal laser scanning micrographs of vibratome sections. Dorsal is up in all images and anterior is left in (B,G) . Er-PDF-I-ir (magenta), Er-PDF-II-ir (green) and DNA (gray). (A,B) Er-PDF-I and Er-PDF-II are co-localized in neuropil and somata, albeit at different intensities. (C) Somata (arrowheads) and processes (arrows) of dorsal cell group exhibit equal intensity levels of Er-PDF-I-ir and Er-PDF-II-ir. (D) In contrast, somata and processes of ventral cell group occur in three variants: (i) Er-PDF-I-ir and Er-PDF-II-ir at equal levels (open arrowheads), (ii) Er-PDF-I-ir at higher level (arrows), and (iii) Er-PDF-II-ir at higher level (filled arrowheads). (E–H) Differences in expression levels of Er-PDF-I-ir and Er-PDF-I-ir are also seen in optic neuropil (E) , inner lobe (black asterisk) as opposed to remaining lobes of the mushroom bodies (white asterisks) (F) , somata of connecting cords (G) , and somata and neuropil of nerve cords (H) . an, anterior neuropil; ad, anterior division of central body; at, antennal tract; cb, central body; cc, connecting cord; cn, central neuropil; dl, dorsal perikaryal layer; ey, eye; mb, mushroom body; ol, olfactory lobe; on, optic neuropil; ot, optic tract; pd, posterior division of the central body; vl, ventral perikaryal layer; vn, neuropil of ventral nerve cord; vp, perikaryal layer of ventral nerve cord. Scale bars: 50 μm (A,B) and 20 μm (C–H) .

    Journal: Frontiers in Endocrinology

    Article Title: Analysis of Pigment-Dispersing Factor Neuropeptides and Their Receptor in a Velvet Worm

    doi: 10.3389/fendo.2020.00273

    Figure Lengend Snippet: Combined immunolocalization of Er-PDF-I and Er-PDF-I in the brain of E. rowelli . Confocal laser scanning micrographs of vibratome sections. Dorsal is up in all images and anterior is left in (B,G) . Er-PDF-I-ir (magenta), Er-PDF-II-ir (green) and DNA (gray). (A,B) Er-PDF-I and Er-PDF-II are co-localized in neuropil and somata, albeit at different intensities. (C) Somata (arrowheads) and processes (arrows) of dorsal cell group exhibit equal intensity levels of Er-PDF-I-ir and Er-PDF-II-ir. (D) In contrast, somata and processes of ventral cell group occur in three variants: (i) Er-PDF-I-ir and Er-PDF-II-ir at equal levels (open arrowheads), (ii) Er-PDF-I-ir at higher level (arrows), and (iii) Er-PDF-II-ir at higher level (filled arrowheads). (E–H) Differences in expression levels of Er-PDF-I-ir and Er-PDF-I-ir are also seen in optic neuropil (E) , inner lobe (black asterisk) as opposed to remaining lobes of the mushroom bodies (white asterisks) (F) , somata of connecting cords (G) , and somata and neuropil of nerve cords (H) . an, anterior neuropil; ad, anterior division of central body; at, antennal tract; cb, central body; cc, connecting cord; cn, central neuropil; dl, dorsal perikaryal layer; ey, eye; mb, mushroom body; ol, olfactory lobe; on, optic neuropil; ot, optic tract; pd, posterior division of the central body; vl, ventral perikaryal layer; vn, neuropil of ventral nerve cord; vp, perikaryal layer of ventral nerve cord. Scale bars: 50 μm (A,B) and 20 μm (C–H) .

    Article Snippet: Confocal Laser Scanning Microscopy and Image ProcessingWhole mount preparations and vibratome sections were analyzed using a confocal laser scanning microscope (Zeiss LSM 880; Carl Zeiss Microscopy GmbH, Jena, Germany) equipped with an Airyscan module.

    Techniques: Expressing

    Combined immunolocalization of Er-PDF-II and Er-PDFR in E. rowelli . Confocal laser scanning micrographs of vibratome sections. Dorsal is up in all images. Er-PDF-II (yellow), Er-PDFR (cyan), and DNA (gray). Note that cuticle is autofluorescent. (A) Overview of protocerebrum. (B) Detailed view of dorsal perikaryal layer. (C) Detailed view of ventral perikaryal layer. Note that Er-PDF-II and Er-PDFR are co-localized in some cells of the ventral group (arrowheads). at, antennal tract; cn, central neuropil; dl, dorsal perikaryal layer; ey, eye; mb, mushroom body; ol, olfactory lobe; on, optic neuropil; vl, ventral perikaryal layer; Scale bars: 50 μm (A) and 10 μm (B,C) .

    Journal: Frontiers in Endocrinology

    Article Title: Analysis of Pigment-Dispersing Factor Neuropeptides and Their Receptor in a Velvet Worm

    doi: 10.3389/fendo.2020.00273

    Figure Lengend Snippet: Combined immunolocalization of Er-PDF-II and Er-PDFR in E. rowelli . Confocal laser scanning micrographs of vibratome sections. Dorsal is up in all images. Er-PDF-II (yellow), Er-PDFR (cyan), and DNA (gray). Note that cuticle is autofluorescent. (A) Overview of protocerebrum. (B) Detailed view of dorsal perikaryal layer. (C) Detailed view of ventral perikaryal layer. Note that Er-PDF-II and Er-PDFR are co-localized in some cells of the ventral group (arrowheads). at, antennal tract; cn, central neuropil; dl, dorsal perikaryal layer; ey, eye; mb, mushroom body; ol, olfactory lobe; on, optic neuropil; vl, ventral perikaryal layer; Scale bars: 50 μm (A) and 10 μm (B,C) .

    Article Snippet: Confocal Laser Scanning Microscopy and Image ProcessingWhole mount preparations and vibratome sections were analyzed using a confocal laser scanning microscope (Zeiss LSM 880; Carl Zeiss Microscopy GmbH, Jena, Germany) equipped with an Airyscan module.

    Techniques:

    Combined immunolocalization of Er-PDF-I and Er-PDF-II in the brain of E. rowelli . Maximum projection of a substack of confocal laser scanning micrographs. Dotted lines indicate outline of brain. Anterior is up in all images. Er-PDF-I-ir (magenta), Er-PDF-II-ir (green), and co-localization (white). (A,B) Dorsal Er-PDF-I-ir /Er-PDF-II-ir cell group is subdivided in anterior (filled arrowheads) and median cell groups (open arrowheads). Ventrally located somata with strong Er-PDF-I immunoreactivity occur anteriorly and laterally (A ′ ,C ′ ) , whereas those with strong Er-PDF-II immunoreactivity are located further posteriorly (B ′ ,C ′ ) . (C) Note that all Er-PDF-I-ir and Er-PDF-II-ir somata of dorsal group appear in white, indicating co-localization of both peptides at similar levels. Scale bar: 50 μm.

    Journal: Frontiers in Endocrinology

    Article Title: Analysis of Pigment-Dispersing Factor Neuropeptides and Their Receptor in a Velvet Worm

    doi: 10.3389/fendo.2020.00273

    Figure Lengend Snippet: Combined immunolocalization of Er-PDF-I and Er-PDF-II in the brain of E. rowelli . Maximum projection of a substack of confocal laser scanning micrographs. Dotted lines indicate outline of brain. Anterior is up in all images. Er-PDF-I-ir (magenta), Er-PDF-II-ir (green), and co-localization (white). (A,B) Dorsal Er-PDF-I-ir /Er-PDF-II-ir cell group is subdivided in anterior (filled arrowheads) and median cell groups (open arrowheads). Ventrally located somata with strong Er-PDF-I immunoreactivity occur anteriorly and laterally (A ′ ,C ′ ) , whereas those with strong Er-PDF-II immunoreactivity are located further posteriorly (B ′ ,C ′ ) . (C) Note that all Er-PDF-I-ir and Er-PDF-II-ir somata of dorsal group appear in white, indicating co-localization of both peptides at similar levels. Scale bar: 50 μm.

    Article Snippet: Confocal Laser Scanning Microscopy and Image ProcessingWhole mount preparations and vibratome sections were analyzed using a confocal laser scanning microscope (Zeiss LSM 880; Carl Zeiss Microscopy GmbH, Jena, Germany) equipped with an Airyscan module.

    Techniques:

    Combined immunolocalization of either Er-PDF-I or Er-PDF-I with Er-PDFR in the vascular system of E. rowelli . Confocal laser scanning micrographs of vibratome sections. Anterior is left in all images. Er-PDF-I (magenta), Er-PDF-II (green), Er-PDFR (cyan), and DNA (gray). (A) Er-PDF-I and Er-PDF-II are co-localized in heart nerve with varying intensities, especially within fine branches (arrowheads). (B,C) PDFR immunoreactivity in cell membranes of hemocytes found in heart lumen (B) and body cavity (C) . he, hemocyte; hn, heart nerve. Scale bars: 25 μm (A) , 10 μm (B) , and 5 μm (C) .

    Journal: Frontiers in Endocrinology

    Article Title: Analysis of Pigment-Dispersing Factor Neuropeptides and Their Receptor in a Velvet Worm

    doi: 10.3389/fendo.2020.00273

    Figure Lengend Snippet: Combined immunolocalization of either Er-PDF-I or Er-PDF-I with Er-PDFR in the vascular system of E. rowelli . Confocal laser scanning micrographs of vibratome sections. Anterior is left in all images. Er-PDF-I (magenta), Er-PDF-II (green), Er-PDFR (cyan), and DNA (gray). (A) Er-PDF-I and Er-PDF-II are co-localized in heart nerve with varying intensities, especially within fine branches (arrowheads). (B,C) PDFR immunoreactivity in cell membranes of hemocytes found in heart lumen (B) and body cavity (C) . he, hemocyte; hn, heart nerve. Scale bars: 25 μm (A) , 10 μm (B) , and 5 μm (C) .

    Article Snippet: Confocal Laser Scanning Microscopy and Image ProcessingWhole mount preparations and vibratome sections were analyzed using a confocal laser scanning microscope (Zeiss LSM 880; Carl Zeiss Microscopy GmbH, Jena, Germany) equipped with an Airyscan module.

    Techniques:

    Combined immunolocalization of either Er-PDF-I or Er-PDF-I with Er-PDFR in the visual system E. rowelli . Confocal laser scanning micrographs of vibratome sections. Dorsal is up in all images. Er-PDF-I (magenta), Er-PDF-II (yellow), Er-PDFR (cyan) and DNA (gray). (A–D) PDFR immunoreactivity occurs in optic neuropil, few somata of optic ganglion (arrowheads), rhabdomeric layer ( A–C and insets for detail), and pigment granules (C,D) . Square in (C) indicates region of (D) . Note the lack of co-localization od Er-PDFR with Er-PDF-I (A) or ER-PDF-II (B) . on, optic neuropil; pe, perikaryal layer of eye; pg, pigment granules rh, rhabdomeric layer; layer. Scale bars: 20 μm (A–C) and 2 μm (insets of A , B,D ).

    Journal: Frontiers in Endocrinology

    Article Title: Analysis of Pigment-Dispersing Factor Neuropeptides and Their Receptor in a Velvet Worm

    doi: 10.3389/fendo.2020.00273

    Figure Lengend Snippet: Combined immunolocalization of either Er-PDF-I or Er-PDF-I with Er-PDFR in the visual system E. rowelli . Confocal laser scanning micrographs of vibratome sections. Dorsal is up in all images. Er-PDF-I (magenta), Er-PDF-II (yellow), Er-PDFR (cyan) and DNA (gray). (A–D) PDFR immunoreactivity occurs in optic neuropil, few somata of optic ganglion (arrowheads), rhabdomeric layer ( A–C and insets for detail), and pigment granules (C,D) . Square in (C) indicates region of (D) . Note the lack of co-localization od Er-PDFR with Er-PDF-I (A) or ER-PDF-II (B) . on, optic neuropil; pe, perikaryal layer of eye; pg, pigment granules rh, rhabdomeric layer; layer. Scale bars: 20 μm (A–C) and 2 μm (insets of A , B,D ).

    Article Snippet: Confocal Laser Scanning Microscopy and Image ProcessingWhole mount preparations and vibratome sections were analyzed using a confocal laser scanning microscope (Zeiss LSM 880; Carl Zeiss Microscopy GmbH, Jena, Germany) equipped with an Airyscan module.

    Techniques:

    Immunolocalization of Er-PDF-I and Er-PDF-II in E. rowelli . Confocal laser scanning micrographs of vibratome sections. Dorsal is up in all images. Hatched line indicates median regions. Er-PDF-I-ir (magenta), Er-PDF-II-ir (green) and DNA-labeling (gray) from anterior to posterior through head (A–C) and trunk (D) . Note similar distribution of either peptide in brain and ventral nerve cords. (A) Arrows indicate dorsal groups of somata in protocerebrum. Insets show large varicosities in PDF-immunoreactive fibers. (B) Arrowheads point to large ventral groups of somata in protocerebrum. Asterisks indicate four lobes of mushroom bodies. (C) Filled arrowheads point to somata in deutocerebrum. Empty arrowheads demarcate somata in connecting cords. (D) Cross sections of ventral nerve cords. at, antennal tract; cb, central body; cc, connecting cord; cn, central neuropil; dc, deutocerebrum; dl, dorsal perikaryal layer; ol, olfactory lobe; on, optic neuropil; ot, optic tract; vl, ventral perikaryal layer; vn, neuropil of ventral nerve cord. Scale bars: 50 μm (A–D) and 500 nm (insets).

    Journal: Frontiers in Endocrinology

    Article Title: Analysis of Pigment-Dispersing Factor Neuropeptides and Their Receptor in a Velvet Worm

    doi: 10.3389/fendo.2020.00273

    Figure Lengend Snippet: Immunolocalization of Er-PDF-I and Er-PDF-II in E. rowelli . Confocal laser scanning micrographs of vibratome sections. Dorsal is up in all images. Hatched line indicates median regions. Er-PDF-I-ir (magenta), Er-PDF-II-ir (green) and DNA-labeling (gray) from anterior to posterior through head (A–C) and trunk (D) . Note similar distribution of either peptide in brain and ventral nerve cords. (A) Arrows indicate dorsal groups of somata in protocerebrum. Insets show large varicosities in PDF-immunoreactive fibers. (B) Arrowheads point to large ventral groups of somata in protocerebrum. Asterisks indicate four lobes of mushroom bodies. (C) Filled arrowheads point to somata in deutocerebrum. Empty arrowheads demarcate somata in connecting cords. (D) Cross sections of ventral nerve cords. at, antennal tract; cb, central body; cc, connecting cord; cn, central neuropil; dc, deutocerebrum; dl, dorsal perikaryal layer; ol, olfactory lobe; on, optic neuropil; ot, optic tract; vl, ventral perikaryal layer; vn, neuropil of ventral nerve cord. Scale bars: 50 μm (A–D) and 500 nm (insets).

    Article Snippet: Confocal Laser Scanning Microscopy and Image ProcessingWhole mount preparations and vibratome sections were analyzed using a confocal laser scanning microscope (Zeiss LSM 880; Carl Zeiss Microscopy GmbH, Jena, Germany) equipped with an Airyscan module.

    Techniques: DNA Labeling

    Combined immunolocalization of Er-PDF-I and Er-PDFR in E. rowelli . Confocal laser scanning micrographs of vibratome sections. Dorsal is up in all images. Er-PDF-I (magenta), Er-PDFR (cyan), and DNA (gray). Note that cuticle is autofluorescent. (A) Overview of protocerebrum. (B) Detailed view of dorsal perikaryal layer. (C) Detailed view of ventral perikaryal layer. Note that Er-PDF-I and Er-PDFR are co-localized only in some cells of ventral group (arrowheads). at, antennal tract; cn, central neuropil; dl, dorsal perikaryal layer; ey, eye; mb, mushroom body; ol, olfactory lobe; on, optic neuropil; vl, ventral perikaryal layer; Scale bars: 50 μm (A) and 10 μm (B,C) .

    Journal: Frontiers in Endocrinology

    Article Title: Analysis of Pigment-Dispersing Factor Neuropeptides and Their Receptor in a Velvet Worm

    doi: 10.3389/fendo.2020.00273

    Figure Lengend Snippet: Combined immunolocalization of Er-PDF-I and Er-PDFR in E. rowelli . Confocal laser scanning micrographs of vibratome sections. Dorsal is up in all images. Er-PDF-I (magenta), Er-PDFR (cyan), and DNA (gray). Note that cuticle is autofluorescent. (A) Overview of protocerebrum. (B) Detailed view of dorsal perikaryal layer. (C) Detailed view of ventral perikaryal layer. Note that Er-PDF-I and Er-PDFR are co-localized only in some cells of ventral group (arrowheads). at, antennal tract; cn, central neuropil; dl, dorsal perikaryal layer; ey, eye; mb, mushroom body; ol, olfactory lobe; on, optic neuropil; vl, ventral perikaryal layer; Scale bars: 50 μm (A) and 10 μm (B,C) .

    Article Snippet: Confocal Laser Scanning Microscopy and Image ProcessingWhole mount preparations and vibratome sections were analyzed using a confocal laser scanning microscope (Zeiss LSM 880; Carl Zeiss Microscopy GmbH, Jena, Germany) equipped with an Airyscan module.

    Techniques:

    RepA-dependent nucleolar exclusion of V2. RFP-H2B plant leaves were infiltrated with Agrobacterium tumefaciens cultures carrying constructs to express GFP-V2 and the other six individual ORFs (V1, V3, V4, V5, RepA, and Rep) of MMDaV. Fluorescence was visualized under a Zeiss LSM 880 confocal laser scanning microscope at 36 hpi. The corresponding region in the white box in column 1 is magnified and shown from Column 2 to Column 5. RFP-H2B was used as a nuclear marker. This experiment was done three times and more than 20 cells were observed per sample and replicate. Scale bars correspond to 10 μm.

    Journal: Frontiers in Microbiology

    Article Title: RepA Promotes the Nucleolar Exclusion of the V2 Protein of Mulberry Mosaic Dwarf-Associated Virus

    doi: 10.3389/fmicb.2020.01828

    Figure Lengend Snippet: RepA-dependent nucleolar exclusion of V2. RFP-H2B plant leaves were infiltrated with Agrobacterium tumefaciens cultures carrying constructs to express GFP-V2 and the other six individual ORFs (V1, V3, V4, V5, RepA, and Rep) of MMDaV. Fluorescence was visualized under a Zeiss LSM 880 confocal laser scanning microscope at 36 hpi. The corresponding region in the white box in column 1 is magnified and shown from Column 2 to Column 5. RFP-H2B was used as a nuclear marker. This experiment was done three times and more than 20 cells were observed per sample and replicate. Scale bars correspond to 10 μm.

    Article Snippet: Confocal Microscopy Imaging of fluorescent proteins in the epidermal cells of agroinfiltrated N. benthamiana or RFP-H2B plants was performed on a laser-scanning confocal microscope (LSM880; Carl Zeiss, Jena, Germany) at 36–48 hours post-infiltration (hpi).

    Techniques: Construct, Fluorescence, Laser-Scanning Microscopy, Marker

    V2 interacts with NbFib2. (A) Yeast two-hybrid assay showing the interaction between V2 and NbFib2 in yeast cells. Full-length NbFib2 was expressed as GAL4 DNA-binding domain fusion (BD, bait) and V2 was expressed as GAL4 activation domain fusion (AD, prey) in yeast cells of the strain Y2H Gold. The interaction of p53 and T was used as a positive control, and cotransformation of Lam and T was used as a negative control. Growth on the plates lacking leucine and tryptophan (SD/-LT) indicates successful transformation of both prey and bait vectors, respectively. Interaction between NbFib2 and V2 is indicated by growth of yeast cells on media also lacking histidine supplementing with 5 mM 3-amino-1,2,4-triazole (SD/-LTH + 3-AT). (B) Bimolecular fluorescence complementation assay (BiFC) assay showing the interaction between NbFib2 and V2 in plant cells. Constructs containing N-terminal YFP fusion (nYFP) and C-terminal YFP fusion (cYFP) fusions were infiltrated into RFP-H2B plant leaves. Combinations of BiFC constructs are shown at the top of each panel. Images were taken using a Zeiss LSM 880 confocal laser scanning microscope at 48 hpi. Reconstituted YFP signals resulting from V2-NbFib2 interaction are displayed as a false-green color. RFP-H2B served as a nuclear marker. Note that deletion of the predicted nuclear localization signal (from amino acid 61–76) of V2 abolishes its interaction with NbFib2. (C) Colocalization analysis of NbFib2 with V2 and V2 mutant in the epidermal cells of N. benthamiana by Zeiss LSM 880 confocal laser scanning microscope at 36 hpi. At least 60 cells from three repeats were examined. Scale bars correspond to 10 μm. (D) Immunoblot of proteins from RFP-H2B plant leaves infiltrated with construct as indicated using anti-GFP antibody. Ponceau staining of the large subunit of Rubisco serves as a loading control.

    Journal: Frontiers in Microbiology

    Article Title: RepA Promotes the Nucleolar Exclusion of the V2 Protein of Mulberry Mosaic Dwarf-Associated Virus

    doi: 10.3389/fmicb.2020.01828

    Figure Lengend Snippet: V2 interacts with NbFib2. (A) Yeast two-hybrid assay showing the interaction between V2 and NbFib2 in yeast cells. Full-length NbFib2 was expressed as GAL4 DNA-binding domain fusion (BD, bait) and V2 was expressed as GAL4 activation domain fusion (AD, prey) in yeast cells of the strain Y2H Gold. The interaction of p53 and T was used as a positive control, and cotransformation of Lam and T was used as a negative control. Growth on the plates lacking leucine and tryptophan (SD/-LT) indicates successful transformation of both prey and bait vectors, respectively. Interaction between NbFib2 and V2 is indicated by growth of yeast cells on media also lacking histidine supplementing with 5 mM 3-amino-1,2,4-triazole (SD/-LTH + 3-AT). (B) Bimolecular fluorescence complementation assay (BiFC) assay showing the interaction between NbFib2 and V2 in plant cells. Constructs containing N-terminal YFP fusion (nYFP) and C-terminal YFP fusion (cYFP) fusions were infiltrated into RFP-H2B plant leaves. Combinations of BiFC constructs are shown at the top of each panel. Images were taken using a Zeiss LSM 880 confocal laser scanning microscope at 48 hpi. Reconstituted YFP signals resulting from V2-NbFib2 interaction are displayed as a false-green color. RFP-H2B served as a nuclear marker. Note that deletion of the predicted nuclear localization signal (from amino acid 61–76) of V2 abolishes its interaction with NbFib2. (C) Colocalization analysis of NbFib2 with V2 and V2 mutant in the epidermal cells of N. benthamiana by Zeiss LSM 880 confocal laser scanning microscope at 36 hpi. At least 60 cells from three repeats were examined. Scale bars correspond to 10 μm. (D) Immunoblot of proteins from RFP-H2B plant leaves infiltrated with construct as indicated using anti-GFP antibody. Ponceau staining of the large subunit of Rubisco serves as a loading control.

    Article Snippet: Confocal Microscopy Imaging of fluorescent proteins in the epidermal cells of agroinfiltrated N. benthamiana or RFP-H2B plants was performed on a laser-scanning confocal microscope (LSM880; Carl Zeiss, Jena, Germany) at 36–48 hours post-infiltration (hpi).

    Techniques: Y2H Assay, Binding Assay, Activation Assay, Positive Control, Laser Capture Microdissection, Negative Control, Transformation Assay, Bimolecular Fluorescence Complementation Assay, Construct, Laser-Scanning Microscopy, Marker, Mutagenesis, Staining

    RepA interacts with V2 in yeast and plant cells. (A) Yeast two-hybrid assay showing the interaction between RepA and V2 in yeast cells. Growth of yeast cotransformants containing the BD-RepA and AD-V2 fusions on the plates lacking leucine, tryptophan, histidine, and adenine (SD/-LTHA) indicates specific interaction between RepA and V2. (B) BiFC assay showing the interaction between RepA and V2 in plant cells. Combinations of BiFC constructs are shown at the top of each panel. Images were taken using a Zeiss LSM 880 confocal laser scanning microscope at 48 hpi. Reconstituted YFP signals as a consequence of V2-RepA interaction are depicted as a false-green color. RFP-H2B served as a nuclear marker.

    Journal: Frontiers in Microbiology

    Article Title: RepA Promotes the Nucleolar Exclusion of the V2 Protein of Mulberry Mosaic Dwarf-Associated Virus

    doi: 10.3389/fmicb.2020.01828

    Figure Lengend Snippet: RepA interacts with V2 in yeast and plant cells. (A) Yeast two-hybrid assay showing the interaction between RepA and V2 in yeast cells. Growth of yeast cotransformants containing the BD-RepA and AD-V2 fusions on the plates lacking leucine, tryptophan, histidine, and adenine (SD/-LTHA) indicates specific interaction between RepA and V2. (B) BiFC assay showing the interaction between RepA and V2 in plant cells. Combinations of BiFC constructs are shown at the top of each panel. Images were taken using a Zeiss LSM 880 confocal laser scanning microscope at 48 hpi. Reconstituted YFP signals as a consequence of V2-RepA interaction are depicted as a false-green color. RFP-H2B served as a nuclear marker.

    Article Snippet: Confocal Microscopy Imaging of fluorescent proteins in the epidermal cells of agroinfiltrated N. benthamiana or RFP-H2B plants was performed on a laser-scanning confocal microscope (LSM880; Carl Zeiss, Jena, Germany) at 36–48 hours post-infiltration (hpi).

    Techniques: Y2H Assay, Bimolecular Fluorescence Complementation Assay, Construct, Laser-Scanning Microscopy, Marker

    V2 localization within the nucleolus is modulated in the presence of mulberry mosaic dwarf-associated geminivirus (MMDaV). (A) Subcellular localization of green fluorescent protein (GFP) or GFP-V2 fusion in the absence or presence of MMDaV infection in transgenic Nicotiana benthamian a plants expressing red fluorescent protein (RFP)-tagged histone 2B (RFP-H2B). RFP-H2B was used as a nuclear marker. (B) Colocalization analysis of V2 and fibrillarin 2 (NbFib2) in the absence or presence of MMDaV in N. benthamiana plants. To create an environment mimicking MMDaV infection, the infectious clone of MMDaV was infiltrated into RFP-H2B or N. benthamiana leaves 12 h prior to the infiltration of GFP or GFP-V2. Images were taken using Zeiss LSM 880 confocal laser scanning microscope at 36 hours post infiltration (hpi) of GFP or GFP-V2. This experiment was done three times and more than 20 cells were observed per sample and replicate. A representative image is shown for each set. The corresponding region in the white box in column 1 is magnified and shown from Column 2 to Column 5. Scale bars correspond to 10 μm.

    Journal: Frontiers in Microbiology

    Article Title: RepA Promotes the Nucleolar Exclusion of the V2 Protein of Mulberry Mosaic Dwarf-Associated Virus

    doi: 10.3389/fmicb.2020.01828

    Figure Lengend Snippet: V2 localization within the nucleolus is modulated in the presence of mulberry mosaic dwarf-associated geminivirus (MMDaV). (A) Subcellular localization of green fluorescent protein (GFP) or GFP-V2 fusion in the absence or presence of MMDaV infection in transgenic Nicotiana benthamian a plants expressing red fluorescent protein (RFP)-tagged histone 2B (RFP-H2B). RFP-H2B was used as a nuclear marker. (B) Colocalization analysis of V2 and fibrillarin 2 (NbFib2) in the absence or presence of MMDaV in N. benthamiana plants. To create an environment mimicking MMDaV infection, the infectious clone of MMDaV was infiltrated into RFP-H2B or N. benthamiana leaves 12 h prior to the infiltration of GFP or GFP-V2. Images were taken using Zeiss LSM 880 confocal laser scanning microscope at 36 hours post infiltration (hpi) of GFP or GFP-V2. This experiment was done three times and more than 20 cells were observed per sample and replicate. A representative image is shown for each set. The corresponding region in the white box in column 1 is magnified and shown from Column 2 to Column 5. Scale bars correspond to 10 μm.

    Article Snippet: Confocal Microscopy Imaging of fluorescent proteins in the epidermal cells of agroinfiltrated N. benthamiana or RFP-H2B plants was performed on a laser-scanning confocal microscope (LSM880; Carl Zeiss, Jena, Germany) at 36–48 hours post-infiltration (hpi).

    Techniques: Infection, Transgenic Assay, Expressing, Marker, Laser-Scanning Microscopy

    Cathepsin X enables podosome formation. Immature DCs on Day 5 of differentiation were centrifuged with cytospin (Cytofuge) for 6 min at 1000 rpm onto glass cover slides. Actin was labeled with phalloidin-tetramethylrhodamine B isothiocyanate conjugate (500 ng/ml) for 30 min at room temperature. Podosome formation, present in control, immature DCs (A), is prevented by inhibition of cathepsin X during DC differentiation (B). Adhesion of maturing DCs coincides with β 2 -integrin activation and colocalization with actin (C). Immature and mature DCs were labeled by centrifugation with cytospin (Cytofuge), whereas maturing, adherent DCs were labeled by seeding immature DCs onto glass coverslips in 24-well plates in the presence of 20 ng/ml LPS and allowing adherence for 20 h. The active form of β 2 integrin was labeled with mAb 24 (green fluorescence) and colocalized with actin (red fluorescence) in adherent, mature DCs. Meanwhile, formation of typical dendrites in mature DCs (D) was not inhibited by cathepsin X inhibition (E). Original scale bars represent 20 μm. Fluorescence microscopy was performed using a Carl Zeiss LSM 510 confocal microscope. Images were analyzed using Carl Zeiss LSM image software 3.0.

    Journal: Journal of Leukocyte Biology

    Article Title: Maturation of dendritic cells depends on proteolytic cleavage by cathepsin X

    doi: 10.1189/jlb.0508285

    Figure Lengend Snippet: Cathepsin X enables podosome formation. Immature DCs on Day 5 of differentiation were centrifuged with cytospin (Cytofuge) for 6 min at 1000 rpm onto glass cover slides. Actin was labeled with phalloidin-tetramethylrhodamine B isothiocyanate conjugate (500 ng/ml) for 30 min at room temperature. Podosome formation, present in control, immature DCs (A), is prevented by inhibition of cathepsin X during DC differentiation (B). Adhesion of maturing DCs coincides with β 2 -integrin activation and colocalization with actin (C). Immature and mature DCs were labeled by centrifugation with cytospin (Cytofuge), whereas maturing, adherent DCs were labeled by seeding immature DCs onto glass coverslips in 24-well plates in the presence of 20 ng/ml LPS and allowing adherence for 20 h. The active form of β 2 integrin was labeled with mAb 24 (green fluorescence) and colocalized with actin (red fluorescence) in adherent, mature DCs. Meanwhile, formation of typical dendrites in mature DCs (D) was not inhibited by cathepsin X inhibition (E). Original scale bars represent 20 μm. Fluorescence microscopy was performed using a Carl Zeiss LSM 510 confocal microscope. Images were analyzed using Carl Zeiss LSM image software 3.0.

    Article Snippet: Fluorescence microscopy was performed using a Carl Zeiss LSM 510 confocal microscope.

    Techniques: Labeling, Inhibition, Activation Assay, Centrifugation, Fluorescence, Microscopy, Software

    Phenotypic characteristics of DC maturation. Surface marker expression was determined by FACS analysis of mature DCs stimulated for 48 h with LPS (20 ng/ml; A). Broken line shows staining with an isotype control, dotted line the staining of mature DCs, and solid line staining of DCs matured in the presence of the cathepsin X inhibitor. The results are representative of three independent experiments, and the average MFI for control and mAb 2F12-treated DCs is given in the right top corner in histograms. Surface expression of cathepsin X (solid line) was evaluated in adherent maturing and floating mature DCs (B and C). Immature DCs were stimulated with 20 ng/ml LPS for 48 h or TNF-α for 5 days and analyzed for cathepsin X (katX) membrane (nonpermeabilized, DC) or intracellular (permeabilized, DC) expression. Broken lines represent isotype controls (B). Confocal images of cathepsin X translocation to the plasma membrane in maturing, adherent DCs (C). Original scale bars represent 20 μm. Fluorescence microscopy was performed using a Carl Zeiss LSM 510 confocal microscope. Images were analyzed using Carl Zeiss LSM image software 3.0.

    Journal: Journal of Leukocyte Biology

    Article Title: Maturation of dendritic cells depends on proteolytic cleavage by cathepsin X

    doi: 10.1189/jlb.0508285

    Figure Lengend Snippet: Phenotypic characteristics of DC maturation. Surface marker expression was determined by FACS analysis of mature DCs stimulated for 48 h with LPS (20 ng/ml; A). Broken line shows staining with an isotype control, dotted line the staining of mature DCs, and solid line staining of DCs matured in the presence of the cathepsin X inhibitor. The results are representative of three independent experiments, and the average MFI for control and mAb 2F12-treated DCs is given in the right top corner in histograms. Surface expression of cathepsin X (solid line) was evaluated in adherent maturing and floating mature DCs (B and C). Immature DCs were stimulated with 20 ng/ml LPS for 48 h or TNF-α for 5 days and analyzed for cathepsin X (katX) membrane (nonpermeabilized, DC) or intracellular (permeabilized, DC) expression. Broken lines represent isotype controls (B). Confocal images of cathepsin X translocation to the plasma membrane in maturing, adherent DCs (C). Original scale bars represent 20 μm. Fluorescence microscopy was performed using a Carl Zeiss LSM 510 confocal microscope. Images were analyzed using Carl Zeiss LSM image software 3.0.

    Article Snippet: Fluorescence microscopy was performed using a Carl Zeiss LSM 510 confocal microscope.

    Techniques: Marker, Expressing, FACS, Staining, Translocation Assay, Fluorescence, Microscopy, Software

    Cathepsin X colocalizes with the Mac-1 integrin receptor in maturing, adherent DCs. Cathepsin X was labeled with Alexa Fluor 488-labeled mouse 2F12 mAb that recognizes the mature, active form. Absence of colocalization of cathepsin X (green fluorescence) and Mac-1 integrin receptor (red fluorescence) in immature DC (iDC; A) and mature DCs (C) is shown. Differential interference contrast images are shown, respectively. Original bars, 20 μm. Maturing, adherent DCs were labeled by seeding immature DCs onto glass coverslips in 24-well plates in the presence of 20 ng/ml LPS and allowing adherence for 20 h. The translocation of cathepsin X in maturating, adherent DCs to the plasma membrane, where it colocalizes with the Mac-1 integrin receptor, is demonstrated (B). Original scale bars represent 20 μm. Fluorescence microscopy was performed using a Carl Zeiss LSM 510 confocal microscope. Images were analyzed using Carl Zeiss LSM image software 3.0.

    Journal: Journal of Leukocyte Biology

    Article Title: Maturation of dendritic cells depends on proteolytic cleavage by cathepsin X

    doi: 10.1189/jlb.0508285

    Figure Lengend Snippet: Cathepsin X colocalizes with the Mac-1 integrin receptor in maturing, adherent DCs. Cathepsin X was labeled with Alexa Fluor 488-labeled mouse 2F12 mAb that recognizes the mature, active form. Absence of colocalization of cathepsin X (green fluorescence) and Mac-1 integrin receptor (red fluorescence) in immature DC (iDC; A) and mature DCs (C) is shown. Differential interference contrast images are shown, respectively. Original bars, 20 μm. Maturing, adherent DCs were labeled by seeding immature DCs onto glass coverslips in 24-well plates in the presence of 20 ng/ml LPS and allowing adherence for 20 h. The translocation of cathepsin X in maturating, adherent DCs to the plasma membrane, where it colocalizes with the Mac-1 integrin receptor, is demonstrated (B). Original scale bars represent 20 μm. Fluorescence microscopy was performed using a Carl Zeiss LSM 510 confocal microscope. Images were analyzed using Carl Zeiss LSM image software 3.0.

    Article Snippet: Fluorescence microscopy was performed using a Carl Zeiss LSM 510 confocal microscope.

    Techniques: Labeling, Fluorescence, Translocation Assay, Microscopy, Software

    The expression of wild-type-β-TM EGFP and E41K-β-TM EGFP in C2C12. (A–B) Wild-type β-TM EGFP and (C–D) E41K-β-TM EGFP mutant were transfected in (A and C) the myoblasts and (B and D) myotubes differentiated for three to six days and labeled with TRITC-phalloidin (red) and DAPI (blue) to highlight cell nuclei. WT-β-TM expressed in C2C12 (A) myoblasts or (B) myotubes incorporated well into C2C12 stress fibres (A) and endogenous filamentous actin (B; long arrow) as visualised with phalloidin. The E41K-β-TM mutant appeared to be diffused in transfected myoblasts and intense EGFP aggregates co-localised with endogenous actin in the peripheral area of the cells (C–C”; short open arrows). The E41K-β-TM mutant formed perinuclear aggregates in myotubes that did not show phalloidin labeling (D–D’’; long open arrows). C2C12 cells appeared as fused myoblasts rather than differentiated myotubes in six-day differentiated cultures. Confocal microscopy was performed using a Zeiss LSM 510 Meta confocal microscope or an LSM 700 inverted Axio Observer.Z1 microscope. Scale bar = 10 µm.

    Journal: PLoS ONE

    Article Title: Phenotypes of Myopathy-Related Beta-Tropomyosin Mutants in Human and Mouse Tissue Cultures

    doi: 10.1371/journal.pone.0072396

    Figure Lengend Snippet: The expression of wild-type-β-TM EGFP and E41K-β-TM EGFP in C2C12. (A–B) Wild-type β-TM EGFP and (C–D) E41K-β-TM EGFP mutant were transfected in (A and C) the myoblasts and (B and D) myotubes differentiated for three to six days and labeled with TRITC-phalloidin (red) and DAPI (blue) to highlight cell nuclei. WT-β-TM expressed in C2C12 (A) myoblasts or (B) myotubes incorporated well into C2C12 stress fibres (A) and endogenous filamentous actin (B; long arrow) as visualised with phalloidin. The E41K-β-TM mutant appeared to be diffused in transfected myoblasts and intense EGFP aggregates co-localised with endogenous actin in the peripheral area of the cells (C–C”; short open arrows). The E41K-β-TM mutant formed perinuclear aggregates in myotubes that did not show phalloidin labeling (D–D’’; long open arrows). C2C12 cells appeared as fused myoblasts rather than differentiated myotubes in six-day differentiated cultures. Confocal microscopy was performed using a Zeiss LSM 510 Meta confocal microscope or an LSM 700 inverted Axio Observer.Z1 microscope. Scale bar = 10 µm.

    Article Snippet: Stained cells were imaged using a Zeiss LSM 510 Meta confocal microscope, using 63× oil and 40× objectives, or an LSM 700 inverted Axio Observer.Z1 microscope, using a Plan-Apochromat 63×/1.4 Oil DIC or 40× objective.

    Techniques: Expressing, Mutagenesis, Transfection, Labeling, Confocal Microscopy, Microscopy

    Expression of wild-type β-TM EGFP and E41K-β-TM EGFP in human cells. (A–B) Wild-type β-TM EGFP and (C–D) E41K-β-TM EGFP mutants were transfected in human (A and C) myoblasts and (B and D) myotubes differentiated for three to six days and labeled with TRITC-phalloidin (red) and DAPI (blue) to highlight cell nuclei. (A) WT-β-TM was expressed in human myoblasts or (B) myotubes and incorporated well into endogenous filamentous actin as visualised with phalloidin (long arrows). The E41K-β-TM mutant induced diffuse intranuclear and cytoplasmic clouds in transfected myoblasts (C–Ć ´’’) and perinuclear aggregates in myotubes (D–D´ ´’’; short arrows). Confocal microscopy was performed using a Zeiss LSM 510 Meta confocal microscope or an LSM 700 inverted Axio Observer.Z1 microscope. Scale bar = 10 µm.

    Journal: PLoS ONE

    Article Title: Phenotypes of Myopathy-Related Beta-Tropomyosin Mutants in Human and Mouse Tissue Cultures

    doi: 10.1371/journal.pone.0072396

    Figure Lengend Snippet: Expression of wild-type β-TM EGFP and E41K-β-TM EGFP in human cells. (A–B) Wild-type β-TM EGFP and (C–D) E41K-β-TM EGFP mutants were transfected in human (A and C) myoblasts and (B and D) myotubes differentiated for three to six days and labeled with TRITC-phalloidin (red) and DAPI (blue) to highlight cell nuclei. (A) WT-β-TM was expressed in human myoblasts or (B) myotubes and incorporated well into endogenous filamentous actin as visualised with phalloidin (long arrows). The E41K-β-TM mutant induced diffuse intranuclear and cytoplasmic clouds in transfected myoblasts (C–Ć ´’’) and perinuclear aggregates in myotubes (D–D´ ´’’; short arrows). Confocal microscopy was performed using a Zeiss LSM 510 Meta confocal microscope or an LSM 700 inverted Axio Observer.Z1 microscope. Scale bar = 10 µm.

    Article Snippet: Stained cells were imaged using a Zeiss LSM 510 Meta confocal microscope, using 63× oil and 40× objectives, or an LSM 700 inverted Axio Observer.Z1 microscope, using a Plan-Apochromat 63×/1.4 Oil DIC or 40× objective.

    Techniques: Expressing, Transfection, Labeling, Mutagenesis, Confocal Microscopy, Microscopy

    The expression of E122K and N202K-β-TM EGFP and empty EGFP in C2C12. The cells were labeled with TRITC-phalloidin (red) and DAPI (blue) to highlight cell nuclei. The E122K-β-TM EGFP mutant was transfected in (A) myoblasts and (B) myotubes. (A) The E122K-β-TM mutant was mislocalised and produced intense EGFP aggregates, co-localised with endogenous actin in the peripheral area of both myoblasts (A–A”; short arrows) and differentiated cells (B–B”; short arrows). The E122K mutant induced the perinuclear aggregates in differentiated C2C12 (B and B”; long arrows). C2C12 myoblasts transfected with N202K-β-TM EGFP appeared cytopathic, with a thickened, ruffled cell surface (C–C”; long open arrows). Aggregates in the peripheral (D–D”; short closed arrows) and perinuclear (D–D”; long open arrows) areas of differentiated cells were detected. The transfection of C2C12 myoblasts with empty EGFP vector resulted in the formation of well-organised stress fibres (E–E”). Confocal microscopy was performed using a Zeiss LSM 510 Meta confocal microscope or an LSM 700 inverted Axio Observer.Z1 microscope. Scale bar = 10 µm.

    Journal: PLoS ONE

    Article Title: Phenotypes of Myopathy-Related Beta-Tropomyosin Mutants in Human and Mouse Tissue Cultures

    doi: 10.1371/journal.pone.0072396

    Figure Lengend Snippet: The expression of E122K and N202K-β-TM EGFP and empty EGFP in C2C12. The cells were labeled with TRITC-phalloidin (red) and DAPI (blue) to highlight cell nuclei. The E122K-β-TM EGFP mutant was transfected in (A) myoblasts and (B) myotubes. (A) The E122K-β-TM mutant was mislocalised and produced intense EGFP aggregates, co-localised with endogenous actin in the peripheral area of both myoblasts (A–A”; short arrows) and differentiated cells (B–B”; short arrows). The E122K mutant induced the perinuclear aggregates in differentiated C2C12 (B and B”; long arrows). C2C12 myoblasts transfected with N202K-β-TM EGFP appeared cytopathic, with a thickened, ruffled cell surface (C–C”; long open arrows). Aggregates in the peripheral (D–D”; short closed arrows) and perinuclear (D–D”; long open arrows) areas of differentiated cells were detected. The transfection of C2C12 myoblasts with empty EGFP vector resulted in the formation of well-organised stress fibres (E–E”). Confocal microscopy was performed using a Zeiss LSM 510 Meta confocal microscope or an LSM 700 inverted Axio Observer.Z1 microscope. Scale bar = 10 µm.

    Article Snippet: Stained cells were imaged using a Zeiss LSM 510 Meta confocal microscope, using 63× oil and 40× objectives, or an LSM 700 inverted Axio Observer.Z1 microscope, using a Plan-Apochromat 63×/1.4 Oil DIC or 40× objective.

    Techniques: Expressing, Labeling, Mutagenesis, Transfection, Produced, Plasmid Preparation, Confocal Microscopy, Microscopy

    The expression of K49del and G53ins β-TM-EGFP in C2C12. The K49del β-TM-EGFP mutant was transfected in (A) myoblasts and (B) myotubes and labeled with TRITC-phalloidin (red) and DAPI (blue) to highlight cell nuclei. (A) The K49del-β-TM mutant was mislocalised and showed the diffuse labeling of phalloidin in transfected myoblasts. It induced nuclear and cytoplasmic aggregates (A–A’; long open arrows). The K49del-β-TM mutant produced intense EGFP aggregates, co-localised with endogenous actin in the peripheral area of both myoblasts (A–A”; short arrows) and differentiated cells (B–B”; short open arrows), in addition to perinuclear aggregates in differentiated C2C12 (B and B”; short closed arrows). (B) The K49del-β-TM mutant showed some integration with actin filaments of differentiated C2C12 (B–B”; long arrows). The G53ins mutant appeared mislocalised, showed the diffuse labeling of phalloidin in transfected myoblasts (C–C”) and formed cytoplasmic aggregates (C–C”; long open arrows). Intense aggregates in the peripheral area of both myoblasts (C–C”; short arrows) and differentiated cells (D–D”; short arrows) were observed. Confocal microscopy was performed using a Zeiss LSM 510 Meta confocal microscope or an LSM 700 inverted Axio Observer.Z1 microscope. Scale bar = 10 µm.

    Journal: PLoS ONE

    Article Title: Phenotypes of Myopathy-Related Beta-Tropomyosin Mutants in Human and Mouse Tissue Cultures

    doi: 10.1371/journal.pone.0072396

    Figure Lengend Snippet: The expression of K49del and G53ins β-TM-EGFP in C2C12. The K49del β-TM-EGFP mutant was transfected in (A) myoblasts and (B) myotubes and labeled with TRITC-phalloidin (red) and DAPI (blue) to highlight cell nuclei. (A) The K49del-β-TM mutant was mislocalised and showed the diffuse labeling of phalloidin in transfected myoblasts. It induced nuclear and cytoplasmic aggregates (A–A’; long open arrows). The K49del-β-TM mutant produced intense EGFP aggregates, co-localised with endogenous actin in the peripheral area of both myoblasts (A–A”; short arrows) and differentiated cells (B–B”; short open arrows), in addition to perinuclear aggregates in differentiated C2C12 (B and B”; short closed arrows). (B) The K49del-β-TM mutant showed some integration with actin filaments of differentiated C2C12 (B–B”; long arrows). The G53ins mutant appeared mislocalised, showed the diffuse labeling of phalloidin in transfected myoblasts (C–C”) and formed cytoplasmic aggregates (C–C”; long open arrows). Intense aggregates in the peripheral area of both myoblasts (C–C”; short arrows) and differentiated cells (D–D”; short arrows) were observed. Confocal microscopy was performed using a Zeiss LSM 510 Meta confocal microscope or an LSM 700 inverted Axio Observer.Z1 microscope. Scale bar = 10 µm.

    Article Snippet: Stained cells were imaged using a Zeiss LSM 510 Meta confocal microscope, using 63× oil and 40× objectives, or an LSM 700 inverted Axio Observer.Z1 microscope, using a Plan-Apochromat 63×/1.4 Oil DIC or 40× objective.

    Techniques: Expressing, Mutagenesis, Transfection, Labeling, Produced, Confocal Microscopy, Microscopy

    The expression of E202K-β-TM EGFP and empty EGFP constructs in human cells. The E202K-β-TM EGFP mutant was transfected in human (A–B) myoblasts and (C) myotubes. (D) Empty EGFP construct was transfected in myoblasts. Cells were labeled with TRITC-phalloidin (red) and DAPI (blue) to highlight cell nuclei. The transfection of human myoblasts with mutant N202K-β-TM induced the diffuse cytoplasmic labeling of stress fibres (A–A’; long arrows) and small phalloidin-labeled aggregates in the cytoplasm (A’ and A”; short arrows). Mutant N202K-β-TM formed clouds around the nucleus (B and B”; short arrows), in addition to a well-defined organised filamentous structure of stress fibres (B–B”; long arrows). In many N202K-β-TM EGFP -transfected myotubes, more marked changes in actin structures were observed (C–C”). A large accumulation of mutant N202K-β-TM with the co-localisation of polymerised actin appeared, suggesting the disruption of endogenous actin filaments (C–C”; short arrows). Human myoblasts transfected with empty EGFP vector formed well-organised filamentous structures (D–D”). Confocal microscopy was performed using a Zeiss LSM 510 Meta confocal microscope or an LSM 700 inverted Axio Observer.Z1 microscope. Scale bar = 10 µm.

    Journal: PLoS ONE

    Article Title: Phenotypes of Myopathy-Related Beta-Tropomyosin Mutants in Human and Mouse Tissue Cultures

    doi: 10.1371/journal.pone.0072396

    Figure Lengend Snippet: The expression of E202K-β-TM EGFP and empty EGFP constructs in human cells. The E202K-β-TM EGFP mutant was transfected in human (A–B) myoblasts and (C) myotubes. (D) Empty EGFP construct was transfected in myoblasts. Cells were labeled with TRITC-phalloidin (red) and DAPI (blue) to highlight cell nuclei. The transfection of human myoblasts with mutant N202K-β-TM induced the diffuse cytoplasmic labeling of stress fibres (A–A’; long arrows) and small phalloidin-labeled aggregates in the cytoplasm (A’ and A”; short arrows). Mutant N202K-β-TM formed clouds around the nucleus (B and B”; short arrows), in addition to a well-defined organised filamentous structure of stress fibres (B–B”; long arrows). In many N202K-β-TM EGFP -transfected myotubes, more marked changes in actin structures were observed (C–C”). A large accumulation of mutant N202K-β-TM with the co-localisation of polymerised actin appeared, suggesting the disruption of endogenous actin filaments (C–C”; short arrows). Human myoblasts transfected with empty EGFP vector formed well-organised filamentous structures (D–D”). Confocal microscopy was performed using a Zeiss LSM 510 Meta confocal microscope or an LSM 700 inverted Axio Observer.Z1 microscope. Scale bar = 10 µm.

    Article Snippet: Stained cells were imaged using a Zeiss LSM 510 Meta confocal microscope, using 63× oil and 40× objectives, or an LSM 700 inverted Axio Observer.Z1 microscope, using a Plan-Apochromat 63×/1.4 Oil DIC or 40× objective.

    Techniques: Expressing, Construct, Mutagenesis, Transfection, Labeling, Plasmid Preparation, Confocal Microscopy, Microscopy

    The expression of G53ins-β-TM EGFP in human cells. The G53ins-β-TM EGFP mutant was transfected in human (A) myoblasts and (B) myotubes and labeled with TRITC-phalloidin (red) and DAPI (blue) to highlight cell nuclei. The G53ins-β-TM EGFP mutant produced delocalisation and endogenous actin aggregates in human myoblasts, labeled with phalloidin (A–A”). The cells transfected with the G53ins-β-TM mutant differentiated into myotubes in an advanced, developed state, identified by their elongated shape and multiple nuclei (B–B”). Transfected myotubes showed good integration of the mutant TM into sarcomeric structures (B; long arrow). The G53ins mutant produced diffuse cytoplasmic labeling at the far end of the myotubes (B; short arrow) . Confocal microscopy was performed using a Zeiss LSM 510 Meta confocal microscope or an LSM 700 inverted Axio Observer.Z1 microscope. Scale bar = 10 µm.

    Journal: PLoS ONE

    Article Title: Phenotypes of Myopathy-Related Beta-Tropomyosin Mutants in Human and Mouse Tissue Cultures

    doi: 10.1371/journal.pone.0072396

    Figure Lengend Snippet: The expression of G53ins-β-TM EGFP in human cells. The G53ins-β-TM EGFP mutant was transfected in human (A) myoblasts and (B) myotubes and labeled with TRITC-phalloidin (red) and DAPI (blue) to highlight cell nuclei. The G53ins-β-TM EGFP mutant produced delocalisation and endogenous actin aggregates in human myoblasts, labeled with phalloidin (A–A”). The cells transfected with the G53ins-β-TM mutant differentiated into myotubes in an advanced, developed state, identified by their elongated shape and multiple nuclei (B–B”). Transfected myotubes showed good integration of the mutant TM into sarcomeric structures (B; long arrow). The G53ins mutant produced diffuse cytoplasmic labeling at the far end of the myotubes (B; short arrow) . Confocal microscopy was performed using a Zeiss LSM 510 Meta confocal microscope or an LSM 700 inverted Axio Observer.Z1 microscope. Scale bar = 10 µm.

    Article Snippet: Stained cells were imaged using a Zeiss LSM 510 Meta confocal microscope, using 63× oil and 40× objectives, or an LSM 700 inverted Axio Observer.Z1 microscope, using a Plan-Apochromat 63×/1.4 Oil DIC or 40× objective.

    Techniques: Expressing, Mutagenesis, Transfection, Labeling, Produced, Confocal Microscopy, Microscopy

    The expression of K49del-β-TM EGFP in human cells. (A–D) The K49del-β-TM EGFP mutant was transfected in human myoblasts and (E) myotubes and labeled with TRITC-phalloidin (red) and DAPI (blue) to highlight cell nuclei. The K49del-β-TM co-localised with cytoplasmic and nuclear aggregates of endogenous actin, detectable with phalloidin labeling (A–C”; short arrows). It produced clouds around the nucleus (A and D) but was also incorporated into stress fibres and filamentous lamellipodia (A and A”, B and B”, and D and D”; long arrows). In addition, it induced thickened filamentous structures of endogenous actin (A’–A”; arrow heads). The K49del-β-TM produced small, long rod-shaped intranuclear structures. A subset of aggregates labeled with phalloidin but was not detectably composed of EGFP-tagged mutant K49del-β-TM (D’–D”; short arrows). Frequently, cytoplasmic thickened filamentous structures with no co-localisation of F-actin were found in myoblasts transfected with K49del-β-TM (D and D”; arrow heads). The K49del-β-TM EGFP mutant produced rod-shaped filamentous actin, cytoplasmic aggregates and cloud patterns in human myotubes (E). The rod-shaped structures were labeled with phalloidin, indicating co-localisation with F-actin (E–E”; short arrows). Cytoplasmic thickened filamentous structures were also observed (E and E”; arrow heads). The K49del-β-TM EGFP mutant was also incorporated into filamentous actin (E; long arrow). Confocal microscopy was performed using a Zeiss LSM 510 Meta confocal microscope or an LSM 700 inverted Axio Observer.Z1 microscope. Scale bar = 10 µm.

    Journal: PLoS ONE

    Article Title: Phenotypes of Myopathy-Related Beta-Tropomyosin Mutants in Human and Mouse Tissue Cultures

    doi: 10.1371/journal.pone.0072396

    Figure Lengend Snippet: The expression of K49del-β-TM EGFP in human cells. (A–D) The K49del-β-TM EGFP mutant was transfected in human myoblasts and (E) myotubes and labeled with TRITC-phalloidin (red) and DAPI (blue) to highlight cell nuclei. The K49del-β-TM co-localised with cytoplasmic and nuclear aggregates of endogenous actin, detectable with phalloidin labeling (A–C”; short arrows). It produced clouds around the nucleus (A and D) but was also incorporated into stress fibres and filamentous lamellipodia (A and A”, B and B”, and D and D”; long arrows). In addition, it induced thickened filamentous structures of endogenous actin (A’–A”; arrow heads). The K49del-β-TM produced small, long rod-shaped intranuclear structures. A subset of aggregates labeled with phalloidin but was not detectably composed of EGFP-tagged mutant K49del-β-TM (D’–D”; short arrows). Frequently, cytoplasmic thickened filamentous structures with no co-localisation of F-actin were found in myoblasts transfected with K49del-β-TM (D and D”; arrow heads). The K49del-β-TM EGFP mutant produced rod-shaped filamentous actin, cytoplasmic aggregates and cloud patterns in human myotubes (E). The rod-shaped structures were labeled with phalloidin, indicating co-localisation with F-actin (E–E”; short arrows). Cytoplasmic thickened filamentous structures were also observed (E and E”; arrow heads). The K49del-β-TM EGFP mutant was also incorporated into filamentous actin (E; long arrow). Confocal microscopy was performed using a Zeiss LSM 510 Meta confocal microscope or an LSM 700 inverted Axio Observer.Z1 microscope. Scale bar = 10 µm.

    Article Snippet: Stained cells were imaged using a Zeiss LSM 510 Meta confocal microscope, using 63× oil and 40× objectives, or an LSM 700 inverted Axio Observer.Z1 microscope, using a Plan-Apochromat 63×/1.4 Oil DIC or 40× objective.

    Techniques: Expressing, Mutagenesis, Transfection, Labeling, Produced, Confocal Microscopy, Microscopy

    The expression of E122K-β-TM EGFP in human cells. The E122K-β-TM EGFP mutant was transfected in human (A–C) myoblasts and (D) myotubes and labeled with TRITC-phalloidin (red) and DAPI (blue) to highlight cell nuclei. The E122K-β-TM EGFP mutant formed aggregates of endogenous actin in human myoblasts (A–A”; inset (B–B”)). Moreover, the E122K-β-TM EGFP incorporated in clouds with an unorganised filamentous structure (C–C”). E122K-β-TM EGFP formed intranuclear rods in human myoblasts that were only detectable with phalloidin labeling (C–C”; short arrows). It also integrated well with stress fibres (A–A” and C–C”; long arrows). The transfection of the E122K-β-TM EGFP construct generally resulted in the less well-defined phalloidin labeling of actin filaments at the far end of the transfected myotubes with the appearance of small rod-like structures located at the periphery (D–D”; inset, short arrows). The rod-shaped structures did not label with phalloidin, indicating that they were not accessible to phalloidin or were not composed of filamentous actin. Confocal microscopy was performed using a Zeiss LSM 510 Meta confocal microscope or an LSM 700 inverted Axio Observer.Z1 microscope. Scale bar = 10 µm.

    Journal: PLoS ONE

    Article Title: Phenotypes of Myopathy-Related Beta-Tropomyosin Mutants in Human and Mouse Tissue Cultures

    doi: 10.1371/journal.pone.0072396

    Figure Lengend Snippet: The expression of E122K-β-TM EGFP in human cells. The E122K-β-TM EGFP mutant was transfected in human (A–C) myoblasts and (D) myotubes and labeled with TRITC-phalloidin (red) and DAPI (blue) to highlight cell nuclei. The E122K-β-TM EGFP mutant formed aggregates of endogenous actin in human myoblasts (A–A”; inset (B–B”)). Moreover, the E122K-β-TM EGFP incorporated in clouds with an unorganised filamentous structure (C–C”). E122K-β-TM EGFP formed intranuclear rods in human myoblasts that were only detectable with phalloidin labeling (C–C”; short arrows). It also integrated well with stress fibres (A–A” and C–C”; long arrows). The transfection of the E122K-β-TM EGFP construct generally resulted in the less well-defined phalloidin labeling of actin filaments at the far end of the transfected myotubes with the appearance of small rod-like structures located at the periphery (D–D”; inset, short arrows). The rod-shaped structures did not label with phalloidin, indicating that they were not accessible to phalloidin or were not composed of filamentous actin. Confocal microscopy was performed using a Zeiss LSM 510 Meta confocal microscope or an LSM 700 inverted Axio Observer.Z1 microscope. Scale bar = 10 µm.

    Article Snippet: Stained cells were imaged using a Zeiss LSM 510 Meta confocal microscope, using 63× oil and 40× objectives, or an LSM 700 inverted Axio Observer.Z1 microscope, using a Plan-Apochromat 63×/1.4 Oil DIC or 40× objective.

    Techniques: Expressing, Mutagenesis, Transfection, Labeling, Construct, Confocal Microscopy, Microscopy

    Inactivation of antibody-POD conjugate . Zebrafish embryos at 28 hpf were hybridized with dinitrophenyl-labeled shha and digoxigenin-labeled nkx6.1 RNA probes. (A, B) The expression patterns of shha (A) and nkx6.1 (B) as seen in single-color FISH experiments. In two-color experiments shha transcript was detected first using DyLight633-tyramide and nkx6.1 transcript was detected subsequently by FAM-tyramide. (C-H) Prior to the second round of detection, embryos were incubated for 10 minutes in PBS T (PBS plus 0.1% Tween-20) (C, D), PBS T containing 6% H 2 O 2 (E, F), or 100 mM glycine-HCl pH 2.0 (G, H). Single confocal sections of zebrafish brains are shown in the DyLight633-detection channel (Ch01) and in the FAM-detection channel (Ch02) from a lateral view and with anterior to the left. Images were recorded on a LSM510 microscope (Carl Zeiss) and false colored in ImageJ. Scale bar = 50 μm.

    Journal: Neural Development

    Article Title: Multicolor fluorescent in situ hybridization to define abutting and overlapping gene expression in the embryonic zebrafish brain

    doi: 10.1186/1749-8104-6-10

    Figure Lengend Snippet: Inactivation of antibody-POD conjugate . Zebrafish embryos at 28 hpf were hybridized with dinitrophenyl-labeled shha and digoxigenin-labeled nkx6.1 RNA probes. (A, B) The expression patterns of shha (A) and nkx6.1 (B) as seen in single-color FISH experiments. In two-color experiments shha transcript was detected first using DyLight633-tyramide and nkx6.1 transcript was detected subsequently by FAM-tyramide. (C-H) Prior to the second round of detection, embryos were incubated for 10 minutes in PBS T (PBS plus 0.1% Tween-20) (C, D), PBS T containing 6% H 2 O 2 (E, F), or 100 mM glycine-HCl pH 2.0 (G, H). Single confocal sections of zebrafish brains are shown in the DyLight633-detection channel (Ch01) and in the FAM-detection channel (Ch02) from a lateral view and with anterior to the left. Images were recorded on a LSM510 microscope (Carl Zeiss) and false colored in ImageJ. Scale bar = 50 μm.

    Article Snippet: For pictures of multicolor-fluorescent embryos recorded with a Zeiss LSM510 confocal microscope (Carl Zeiss) embryos were mounted in 75% glycerol in TNTW pH 8 (100 mM Tris pH 8, 150 mM NaCl, 0.1% Tween-20) containing 1% low melting agarose.

    Techniques: Labeling, Expressing, Fluorescence In Situ Hybridization, Incubation, Microscopy

    Three-color FISH . Embryos at 1 dpf were hybridized simultaneously with fluorescein-labeled dbx1a , dinitrophenyl-labeled shha and digoxigenin-labeled dlx2a RNA probes, sequentially detected and visualized using DyLight633-, TAMRA- and FAM-tyramide, respectively. (A-F) In lateral (A-C) and dorsal (D-F) views with anterior to the left, dbx1a , shha and dlx2a expression is shown in green, red and blue, respectively. Overlays of two (A, B, D, E) or all three (C, F) different channels of the same confocal plane are shown. Photographs were taken on a LSM510 confocal microscope (Carl Zeiss). Images were false-colored with RGB look-up tables and processed using ImageJ software. Scale bar = 50 μm. Hy, hypothalamus; M, midbrain; P1, P2, and P3, prosomeres 1, 2, and 3; ZLI, zona limitans intrathalamica.

    Journal: Neural Development

    Article Title: Multicolor fluorescent in situ hybridization to define abutting and overlapping gene expression in the embryonic zebrafish brain

    doi: 10.1186/1749-8104-6-10

    Figure Lengend Snippet: Three-color FISH . Embryos at 1 dpf were hybridized simultaneously with fluorescein-labeled dbx1a , dinitrophenyl-labeled shha and digoxigenin-labeled dlx2a RNA probes, sequentially detected and visualized using DyLight633-, TAMRA- and FAM-tyramide, respectively. (A-F) In lateral (A-C) and dorsal (D-F) views with anterior to the left, dbx1a , shha and dlx2a expression is shown in green, red and blue, respectively. Overlays of two (A, B, D, E) or all three (C, F) different channels of the same confocal plane are shown. Photographs were taken on a LSM510 confocal microscope (Carl Zeiss). Images were false-colored with RGB look-up tables and processed using ImageJ software. Scale bar = 50 μm. Hy, hypothalamus; M, midbrain; P1, P2, and P3, prosomeres 1, 2, and 3; ZLI, zona limitans intrathalamica.

    Article Snippet: For pictures of multicolor-fluorescent embryos recorded with a Zeiss LSM510 confocal microscope (Carl Zeiss) embryos were mounted in 75% glycerol in TNTW pH 8 (100 mM Tris pH 8, 150 mM NaCl, 0.1% Tween-20) containing 1% low melting agarose.

    Techniques: Fluorescence In Situ Hybridization, Labeling, Expressing, Microscopy, Software

    Test for bleed-through between channels using bench-made tyramides . (A-I) Single-color FISH experiments were performed with digoxigenin-labeled dlx2a (A-C), dinitrophenyl-labeled shha (D-F) and fluorescein-labeled dbx1a (G-I) RNA probes and visualized by FAM-, TAMRA- and DyLight633- tyramide, respectively. Lateral views of 1-dpf forebrains are shown with anterior to the left. Only the appropriate detection channel showed a bright signal and no significant bleed-through between channels was observed. Images were recorded with identical settings to those of Figure 7 on a LSM510 microscope (Carl Zeiss) and false colored in ImageJ. Scale bar = 50 μm.

    Journal: Neural Development

    Article Title: Multicolor fluorescent in situ hybridization to define abutting and overlapping gene expression in the embryonic zebrafish brain

    doi: 10.1186/1749-8104-6-10

    Figure Lengend Snippet: Test for bleed-through between channels using bench-made tyramides . (A-I) Single-color FISH experiments were performed with digoxigenin-labeled dlx2a (A-C), dinitrophenyl-labeled shha (D-F) and fluorescein-labeled dbx1a (G-I) RNA probes and visualized by FAM-, TAMRA- and DyLight633- tyramide, respectively. Lateral views of 1-dpf forebrains are shown with anterior to the left. Only the appropriate detection channel showed a bright signal and no significant bleed-through between channels was observed. Images were recorded with identical settings to those of Figure 7 on a LSM510 microscope (Carl Zeiss) and false colored in ImageJ. Scale bar = 50 μm.

    Article Snippet: For pictures of multicolor-fluorescent embryos recorded with a Zeiss LSM510 confocal microscope (Carl Zeiss) embryos were mounted in 75% glycerol in TNTW pH 8 (100 mM Tris pH 8, 150 mM NaCl, 0.1% Tween-20) containing 1% low melting agarose.

    Techniques: Fluorescence In Situ Hybridization, Labeling, Microscopy

    Two-color FISH . (A-D) Dorsal (A, C, D) and lateral (B) views of 1-dpf zebrafish forebrains with anterior to the left. (A, B) Digoxigenin-labeled dbx1a and dinitrophenyl-labeled gsh1 RNA probes are shown in cyan and magenta, respectively. (C, D) Digoxigenin-labeled emx2 and dinitrophenyl-labeled lhx9 RNA probes were visualized in magenta and cyan, respectively. In the telencephalon, emx2 and lhx9 are co-expressed in a lateral cell cluster (yellow). (D) Higher magnification of the area of colocalization (yellow) in the telencephalon. Photographs were taken on a LSM510 confocal microscope (Carl Zeiss). Images were false-colored with RGB look-up-tables and processed using ImageJ software. Scale bar = 50 μm. Di, diencephalon; P1, P2, and P3, prosomeres 1, 2, and 3; Te, telencephalon.

    Journal: Neural Development

    Article Title: Multicolor fluorescent in situ hybridization to define abutting and overlapping gene expression in the embryonic zebrafish brain

    doi: 10.1186/1749-8104-6-10

    Figure Lengend Snippet: Two-color FISH . (A-D) Dorsal (A, C, D) and lateral (B) views of 1-dpf zebrafish forebrains with anterior to the left. (A, B) Digoxigenin-labeled dbx1a and dinitrophenyl-labeled gsh1 RNA probes are shown in cyan and magenta, respectively. (C, D) Digoxigenin-labeled emx2 and dinitrophenyl-labeled lhx9 RNA probes were visualized in magenta and cyan, respectively. In the telencephalon, emx2 and lhx9 are co-expressed in a lateral cell cluster (yellow). (D) Higher magnification of the area of colocalization (yellow) in the telencephalon. Photographs were taken on a LSM510 confocal microscope (Carl Zeiss). Images were false-colored with RGB look-up-tables and processed using ImageJ software. Scale bar = 50 μm. Di, diencephalon; P1, P2, and P3, prosomeres 1, 2, and 3; Te, telencephalon.

    Article Snippet: For pictures of multicolor-fluorescent embryos recorded with a Zeiss LSM510 confocal microscope (Carl Zeiss) embryos were mounted in 75% glycerol in TNTW pH 8 (100 mM Tris pH 8, 150 mM NaCl, 0.1% Tween-20) containing 1% low melting agarose.

    Techniques: Fluorescence In Situ Hybridization, Labeling, Microscopy, Software

    Transduction of immortalized human retinal pigmented epithelial cell line (ARPE-19) with six different rAAV serotypes. ARPE-19 cells were transduced with 10 5 gc/cell of either rAAV1, rAAV2, rAAV5, rAAV7, rAAV8, or rAAV9, as indicated, for 72 h. Infected cells were visualized with rabbit anti-GFP antibodies, and counterstained with DAPI. Control non-transfected cells (NTF) were processed at the same time and in the same fashion. Images are representative of three replicates and were captured using Zeiss LSM510 META confocal microscope at identical exposure settings at 250× magnification. Scale bars = 50 μm.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Recombinant Adeno-Associated Virus Serotype 6 (rAAV6) Potently and Preferentially Transduces Rat Astrocytes In vitro and In vivo

    doi: 10.3389/fncel.2016.00262

    Figure Lengend Snippet: Transduction of immortalized human retinal pigmented epithelial cell line (ARPE-19) with six different rAAV serotypes. ARPE-19 cells were transduced with 10 5 gc/cell of either rAAV1, rAAV2, rAAV5, rAAV7, rAAV8, or rAAV9, as indicated, for 72 h. Infected cells were visualized with rabbit anti-GFP antibodies, and counterstained with DAPI. Control non-transfected cells (NTF) were processed at the same time and in the same fashion. Images are representative of three replicates and were captured using Zeiss LSM510 META confocal microscope at identical exposure settings at 250× magnification. Scale bars = 50 μm.

    Article Snippet: INSETS show matching high magnification images from the same sections, in which GFP fluorescence was captured with a Zeiss LSM510 META confocal microscope at 250× magnification (Scale bars = 50 μm).

    Techniques: Transduction, Infection, Transfection, Microscopy

    Astrocytic tropism of rAAV2 and rAAV6 in the rat cortex. Slices were stained for GFP as a marker of viral transduction (green), the astrocytic marker GFAP (red), and DAPI (blue). White arrows point to a few rAAV2-infected astrocytes (GFP+/GFAP+ cells for rAAV2). Although the vast majority of the AAV6 transduced cells were GFAP-positive, block arrows point to an exception, the rAAV6-transduced neuron-like cell (GFP+/GFAP- cell). Images were taken using a Zeiss LSM510 META confocal microscope at 250× magnification. Scale bars = 50 μm. Images are representative of at least 12 sections from four different rats for each virus.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Recombinant Adeno-Associated Virus Serotype 6 (rAAV6) Potently and Preferentially Transduces Rat Astrocytes In vitro and In vivo

    doi: 10.3389/fncel.2016.00262

    Figure Lengend Snippet: Astrocytic tropism of rAAV2 and rAAV6 in the rat cortex. Slices were stained for GFP as a marker of viral transduction (green), the astrocytic marker GFAP (red), and DAPI (blue). White arrows point to a few rAAV2-infected astrocytes (GFP+/GFAP+ cells for rAAV2). Although the vast majority of the AAV6 transduced cells were GFAP-positive, block arrows point to an exception, the rAAV6-transduced neuron-like cell (GFP+/GFAP- cell). Images were taken using a Zeiss LSM510 META confocal microscope at 250× magnification. Scale bars = 50 μm. Images are representative of at least 12 sections from four different rats for each virus.

    Article Snippet: INSETS show matching high magnification images from the same sections, in which GFP fluorescence was captured with a Zeiss LSM510 META confocal microscope at 250× magnification (Scale bars = 50 μm).

    Techniques: Staining, Marker, Transduction, Infection, Blocking Assay, Microscopy

    Assessment of the ability of rAAV2 and rAAV6 to transduce neurons in the rat cortex. Slices were stained for GFP as a marker of viral transduction (green), the neuronal marker NeuN (red), and DAPI (blue). The bottom right images in each set show overlay of all three signals, with shades of yellow representing colocalization (at this magnification it is not obvious in some cells due to differences in signal intensities and cellular location). Z-stack images were taken using Zeiss LSM510 META confocal microscope at 250× magnification and identical exposure settings. Scale bars = 50 μm. Images are representative of at least 12 sections from four different rats for each virus.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Recombinant Adeno-Associated Virus Serotype 6 (rAAV6) Potently and Preferentially Transduces Rat Astrocytes In vitro and In vivo

    doi: 10.3389/fncel.2016.00262

    Figure Lengend Snippet: Assessment of the ability of rAAV2 and rAAV6 to transduce neurons in the rat cortex. Slices were stained for GFP as a marker of viral transduction (green), the neuronal marker NeuN (red), and DAPI (blue). The bottom right images in each set show overlay of all three signals, with shades of yellow representing colocalization (at this magnification it is not obvious in some cells due to differences in signal intensities and cellular location). Z-stack images were taken using Zeiss LSM510 META confocal microscope at 250× magnification and identical exposure settings. Scale bars = 50 μm. Images are representative of at least 12 sections from four different rats for each virus.

    Article Snippet: INSETS show matching high magnification images from the same sections, in which GFP fluorescence was captured with a Zeiss LSM510 META confocal microscope at 250× magnification (Scale bars = 50 μm).

    Techniques: Transduction, Staining, Marker, Microscopy

    Transduction of primary rat astrocytes with seven different rAAV-green fluorescent protein (GFP) viruses. Astrocytes were transduced with 10 5 gc/cell of either rAAV1, rAAV2, rAAV5, rAAV6, rAAV7, rAAV8, or rAAV9, or treated identically without the addition of virus (non-transfected), for 48 h. Results for rAAV1, rAAV2, rAAV5, and rAAV6 are shown in the top two rows and results for rAAV7, rAAV8, rAAV9 and non-transfected astrocytes are shown in the bottom two rows. Cells were stained with either rabbit or chicken anti-GFP antibodies, as indicated, and counterstained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) to visualize GFP-negative cells. Images were acquired using a confocal Zeiss LSM510 META microscope at 250× magnification, in the same cell preparation with identical exposure settings. Scale bar = 50 μm. Arrowheads point to cells with non-astrocytic morphology that were transduced with rAAV. Results are representative of at least nine wells and four independent experiments per transduction condition.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Recombinant Adeno-Associated Virus Serotype 6 (rAAV6) Potently and Preferentially Transduces Rat Astrocytes In vitro and In vivo

    doi: 10.3389/fncel.2016.00262

    Figure Lengend Snippet: Transduction of primary rat astrocytes with seven different rAAV-green fluorescent protein (GFP) viruses. Astrocytes were transduced with 10 5 gc/cell of either rAAV1, rAAV2, rAAV5, rAAV6, rAAV7, rAAV8, or rAAV9, or treated identically without the addition of virus (non-transfected), for 48 h. Results for rAAV1, rAAV2, rAAV5, and rAAV6 are shown in the top two rows and results for rAAV7, rAAV8, rAAV9 and non-transfected astrocytes are shown in the bottom two rows. Cells were stained with either rabbit or chicken anti-GFP antibodies, as indicated, and counterstained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) to visualize GFP-negative cells. Images were acquired using a confocal Zeiss LSM510 META microscope at 250× magnification, in the same cell preparation with identical exposure settings. Scale bar = 50 μm. Arrowheads point to cells with non-astrocytic morphology that were transduced with rAAV. Results are representative of at least nine wells and four independent experiments per transduction condition.

    Article Snippet: INSETS show matching high magnification images from the same sections, in which GFP fluorescence was captured with a Zeiss LSM510 META confocal microscope at 250× magnification (Scale bars = 50 μm).

    Techniques: Transduction, Transfection, Staining, Microscopy

    Localization of Mel-AF on A375 cells was analyzed using a confocal laser scanning microscope. The cells were treated with FITC-conjugated Mel-AF for 2 h. ( A ) Confocal images show the localization of Mel-AF traced with FITC (green). The membrane compartment was indicated by staining with a specific antibody to CD46, a membrane cofactor protein followed by Alexa Fluor 568 rabbit anti-mouse IgG (red). The cellular nuclei were counterstained by DAPI (blue). ( B ) Orthogonal imaging analysis was performed to confirm the localization of Mel-AF on the cell membrane (yellow). Scale bar, 10 μm.

    Journal: Antibiotics

    Article Title: Melittin from Apis florea Venom as a Promising Therapeutic Agent for Skin Cancer Treatment

    doi: 10.3390/antibiotics9080517

    Figure Lengend Snippet: Localization of Mel-AF on A375 cells was analyzed using a confocal laser scanning microscope. The cells were treated with FITC-conjugated Mel-AF for 2 h. ( A ) Confocal images show the localization of Mel-AF traced with FITC (green). The membrane compartment was indicated by staining with a specific antibody to CD46, a membrane cofactor protein followed by Alexa Fluor 568 rabbit anti-mouse IgG (red). The cellular nuclei were counterstained by DAPI (blue). ( B ) Orthogonal imaging analysis was performed to confirm the localization of Mel-AF on the cell membrane (yellow). Scale bar, 10 μm.

    Article Snippet: Images were acquired using a confocal laser scanning microscope (FluoView FV1000, Olympus).

    Techniques: Laser-Scanning Microscopy, Staining, Imaging

    Effects of recent transfection with anti-EGFP siRNA on SFV at 4 h post infection. CHO cells were transfected with siRNA targeting EGFP mRNA using PF6 (B, C, D) or LF2000 (E, F, G) reagents. Transfected cells were infected with SFV(ZsGreen) 4 at an MOI of 0.1 at 2 h (B, E), 4 h (C, F) or 8 h (D, G) post transfection. All cells were fixed at 4 h post infection. The localization of nsP1 (red) of SFV was revealed using rabbit polyclonal antiserum as the primary detection reagent and Alexa 568 conjugated anti-rabbit antibody as the secondary antibody; ZsGreen was detected by its green fluorescence; the co-localization of these signals is shown as yellow. Nuclei were counter-stained with DAPI (blue). Images were collected using a LSM 710 confocal microscope (Zeiss); scale bar represents 10 µm. A single optical slice from a characteristic infected cell is shown in each panel, and the nuclei of several non-infected cells are also visible. Panel A shows a representative cell from the control experiment (no transfection, cell fixed 4 h after infection with SFV(ZsGreen) 4).

    Journal: PLoS ONE

    Article Title: Transfection of Infectious RNA and DNA/RNA Layered Vectors of Semliki Forest Virus by the Cell-Penetrating Peptide Based Reagent PepFect6

    doi: 10.1371/journal.pone.0069659

    Figure Lengend Snippet: Effects of recent transfection with anti-EGFP siRNA on SFV at 4 h post infection. CHO cells were transfected with siRNA targeting EGFP mRNA using PF6 (B, C, D) or LF2000 (E, F, G) reagents. Transfected cells were infected with SFV(ZsGreen) 4 at an MOI of 0.1 at 2 h (B, E), 4 h (C, F) or 8 h (D, G) post transfection. All cells were fixed at 4 h post infection. The localization of nsP1 (red) of SFV was revealed using rabbit polyclonal antiserum as the primary detection reagent and Alexa 568 conjugated anti-rabbit antibody as the secondary antibody; ZsGreen was detected by its green fluorescence; the co-localization of these signals is shown as yellow. Nuclei were counter-stained with DAPI (blue). Images were collected using a LSM 710 confocal microscope (Zeiss); scale bar represents 10 µm. A single optical slice from a characteristic infected cell is shown in each panel, and the nuclei of several non-infected cells are also visible. Panel A shows a representative cell from the control experiment (no transfection, cell fixed 4 h after infection with SFV(ZsGreen) 4).

    Article Snippet: Images of these samples were collected using LSM 710 confocal microscope (Zeiss).

    Techniques: Transfection, Infection, Fluorescence, Staining, Microscopy

    Prior transfection with anti-EGFP siRNAs does not affect SFV infection at 24 h post transfection. CHO-EGFP cells were transfected with siRNA targeting EGFP mRNA using PF6 (A, B) or LF2000 (C, D) reagents. 24 h post transfection, cells were infected with SFV(ZsGreen) 4 at an MOI of 0.1 and fixed at 4 h (A, C) or 6 h (B, D) post infection. The localization of nsP1 (shown in red) of SFV was revealed using rabbit polyclonal antiserum as the primary detection reagent and Alexa 568 conjugated anti-rabbit antibody as the secondary antibody; ZsGreen was detected by its green fluorescence; the co-localization of these signals in virus replication organelles is shown as yellow. Nuclei were counter-stained with DAPI (blue). Images were collected using a LSM 710 confocal microscope (Zeiss); scale bar represents 10 µm. Each panel shows a single optical slice from one characteristic infected cell, and the nuclei of several non-infected cells are also visible.

    Journal: PLoS ONE

    Article Title: Transfection of Infectious RNA and DNA/RNA Layered Vectors of Semliki Forest Virus by the Cell-Penetrating Peptide Based Reagent PepFect6

    doi: 10.1371/journal.pone.0069659

    Figure Lengend Snippet: Prior transfection with anti-EGFP siRNAs does not affect SFV infection at 24 h post transfection. CHO-EGFP cells were transfected with siRNA targeting EGFP mRNA using PF6 (A, B) or LF2000 (C, D) reagents. 24 h post transfection, cells were infected with SFV(ZsGreen) 4 at an MOI of 0.1 and fixed at 4 h (A, C) or 6 h (B, D) post infection. The localization of nsP1 (shown in red) of SFV was revealed using rabbit polyclonal antiserum as the primary detection reagent and Alexa 568 conjugated anti-rabbit antibody as the secondary antibody; ZsGreen was detected by its green fluorescence; the co-localization of these signals in virus replication organelles is shown as yellow. Nuclei were counter-stained with DAPI (blue). Images were collected using a LSM 710 confocal microscope (Zeiss); scale bar represents 10 µm. Each panel shows a single optical slice from one characteristic infected cell, and the nuclei of several non-infected cells are also visible.

    Article Snippet: Images of these samples were collected using LSM 710 confocal microscope (Zeiss).

    Techniques: Transfection, Infection, Fluorescence, Staining, Microscopy

    Effects of recent transfection with anti-EGFP siRNA on SFV at 6 h post infection. CHO cells were transfected with siRNA targeting EGFP mRNA using PF6 (B, C, D) or LF2000 (E, F, G) reagents. Transfected cells were infected with SFV(ZsGreen) 4 at an MOI of 0.1 at 2 h (B, E), 4 h (C, F) or 8 h (D, G) post transfection. All cells were fixed at 6 h post infection. The localization of nsP1 (red) of SFV was revealed using rabbit polyclonal antiserum as the primary detection reagent and Alexa 568 conjugated anti-rabbit antibody as the secondary antibody; ZsGreen was detected by its green fluorescence; and the co-localization of these signals is shown as yellow. Nuclei were counter-stained with DAPI (blue). Images were collected using a LSM 710 confocal microscope (Zeiss); scale represents 10 µm. Each panel shows a single optical slice from a characteristic infected cell, and the nuclei of several non-infected cells are also visible. Panel A shows a representative cell from the control experiment (no transfection, cell was fixed at 4 h after infection with SFV(ZsGreen) 4).

    Journal: PLoS ONE

    Article Title: Transfection of Infectious RNA and DNA/RNA Layered Vectors of Semliki Forest Virus by the Cell-Penetrating Peptide Based Reagent PepFect6

    doi: 10.1371/journal.pone.0069659

    Figure Lengend Snippet: Effects of recent transfection with anti-EGFP siRNA on SFV at 6 h post infection. CHO cells were transfected with siRNA targeting EGFP mRNA using PF6 (B, C, D) or LF2000 (E, F, G) reagents. Transfected cells were infected with SFV(ZsGreen) 4 at an MOI of 0.1 at 2 h (B, E), 4 h (C, F) or 8 h (D, G) post transfection. All cells were fixed at 6 h post infection. The localization of nsP1 (red) of SFV was revealed using rabbit polyclonal antiserum as the primary detection reagent and Alexa 568 conjugated anti-rabbit antibody as the secondary antibody; ZsGreen was detected by its green fluorescence; and the co-localization of these signals is shown as yellow. Nuclei were counter-stained with DAPI (blue). Images were collected using a LSM 710 confocal microscope (Zeiss); scale represents 10 µm. Each panel shows a single optical slice from a characteristic infected cell, and the nuclei of several non-infected cells are also visible. Panel A shows a representative cell from the control experiment (no transfection, cell was fixed at 4 h after infection with SFV(ZsGreen) 4).

    Article Snippet: Images of these samples were collected using LSM 710 confocal microscope (Zeiss).

    Techniques: Transfection, Infection, Fluorescence, Staining, Microscopy

    CD63 is not localized in the Ct inclusion of infected HEp-2 cells. HEp-2 cells were grown on coverslips and infected with Ct strain D at a MOI of 1 in cell culture medium without antibiotics and cycloheximide. Cells were fixed 44 hr post-infection. In panel A and B, CD63 protein was detected with a mouse monoclonal antibody (red). In panel A, the inclusion membrane was stained with a rabbit antibody against IncA (green), DNA was detected with the Hoechst dye (blue), and the merged image is shown. In panel B, the bacteria were only detected by Hoechst DNA staining and ACBD6 was detected with a rabbit antibody (green), and the merged image is shown. Signal distribution of the orthogonal (z, x) slice of deconvolved Z stack images, indicated by the arrow on the respective merged image, are displayed in the inset of each panels. Images were taken with a Zeiss LSM710 confocal microscope at 43x magnification. The bars in panels represent 10 µm. Nuclei and inclusion are indicated with N and Inc, respectively. Note that CD63 signal was not detected in the inclusions (panel A) and did not co-localize with ABCD6 protein, which was detected inside the inclusions (panel B).

    Journal: PLoS ONE

    Article Title: Eukaryotic Protein Recruitment into the Chlamydia Inclusion: Implications for Survival and Growth

    doi: 10.1371/journal.pone.0036843

    Figure Lengend Snippet: CD63 is not localized in the Ct inclusion of infected HEp-2 cells. HEp-2 cells were grown on coverslips and infected with Ct strain D at a MOI of 1 in cell culture medium without antibiotics and cycloheximide. Cells were fixed 44 hr post-infection. In panel A and B, CD63 protein was detected with a mouse monoclonal antibody (red). In panel A, the inclusion membrane was stained with a rabbit antibody against IncA (green), DNA was detected with the Hoechst dye (blue), and the merged image is shown. In panel B, the bacteria were only detected by Hoechst DNA staining and ACBD6 was detected with a rabbit antibody (green), and the merged image is shown. Signal distribution of the orthogonal (z, x) slice of deconvolved Z stack images, indicated by the arrow on the respective merged image, are displayed in the inset of each panels. Images were taken with a Zeiss LSM710 confocal microscope at 43x magnification. The bars in panels represent 10 µm. Nuclei and inclusion are indicated with N and Inc, respectively. Note that CD63 signal was not detected in the inclusions (panel A) and did not co-localize with ABCD6 protein, which was detected inside the inclusions (panel B).

    Article Snippet: Images were taken with a Zeiss LSM710 confocal microscope at 40x magnification using the same exposure settings for the un-treated and treated coverslips.

    Techniques: Infection, Cell Culture, Staining, Microscopy

    ACBD6 is mobilized into the Ct -inclusion lumen. Cells were grown on coverslips in cell culture medium without antibiotics and cycloheximide. Near confluent cells were fixed in methanol (panel A, B, E and F) or were infected with Ct strain D at a MOI of 1 and fixed 36 hr post-infection (panel C, D, G and H). Results for HeLa cells are shown in panel A to D and those obtained with HEp-2 cells are shown in panel E to H. Human ABCD6 protein was detected with an affinity-purified polyclonal antibody (green). In infected cells, bacterial LPS was detected with a mouse monoclonal antibody (red), and merged images with ACBD6 detection are shown in panel C and G. DNA was stained with the Hoechst dye (blue). In un-infected cells, merged images with stained DNA are shown in panel B and F. In infected cells, merged images with stained DNA of the host and of Ct cells are shown in panel D and H. In un-infected cells, ACBD6 was detected in the nucleus and the cytosol (panel A and E). In infected cells, ACBD6 protein was detected inside Ct inclusions (panel C and G) and not in the nuclei (panel D and H). Images were taken with a Zeiss LSM710 confocal microscope at 63x magnification. The bars in panels represent 10 µm. Representative inclusions are indicated with white arrows and nuclei by a capital N.

    Journal: PLoS ONE

    Article Title: Eukaryotic Protein Recruitment into the Chlamydia Inclusion: Implications for Survival and Growth

    doi: 10.1371/journal.pone.0036843

    Figure Lengend Snippet: ACBD6 is mobilized into the Ct -inclusion lumen. Cells were grown on coverslips in cell culture medium without antibiotics and cycloheximide. Near confluent cells were fixed in methanol (panel A, B, E and F) or were infected with Ct strain D at a MOI of 1 and fixed 36 hr post-infection (panel C, D, G and H). Results for HeLa cells are shown in panel A to D and those obtained with HEp-2 cells are shown in panel E to H. Human ABCD6 protein was detected with an affinity-purified polyclonal antibody (green). In infected cells, bacterial LPS was detected with a mouse monoclonal antibody (red), and merged images with ACBD6 detection are shown in panel C and G. DNA was stained with the Hoechst dye (blue). In un-infected cells, merged images with stained DNA are shown in panel B and F. In infected cells, merged images with stained DNA of the host and of Ct cells are shown in panel D and H. In un-infected cells, ACBD6 was detected in the nucleus and the cytosol (panel A and E). In infected cells, ACBD6 protein was detected inside Ct inclusions (panel C and G) and not in the nuclei (panel D and H). Images were taken with a Zeiss LSM710 confocal microscope at 63x magnification. The bars in panels represent 10 µm. Representative inclusions are indicated with white arrows and nuclei by a capital N.

    Article Snippet: Images were taken with a Zeiss LSM710 confocal microscope at 40x magnification using the same exposure settings for the un-treated and treated coverslips.

    Techniques: Cell Culture, Infection, Affinity Purification, Staining, Microscopy

    ACSL3 but not ACSL6 is recruited into the Ct inclusion. HeLa cells were grown on coverslips and infected with Ct strain D at a MOI of 1 in cell culture medium without antibiotics and cycloheximide. Cells were fixed 36 hr post- infection. DNA was detected with Hoechst 33258 dye (blue). Bacteria were detected with a rabbit antibody against IncA, which was stained with an anti-rabbit antibody labeled with AlexaFluor®488 revealing the inclusion membrane (green, panels A to C) or with a mouse antibody against LPS, which was stained with Cy TM 3-conjugated anti-mouse antibody (red, panel D). Long-chain acyl-CoA synthetase 3, ACSL3, was detected with a mouse monoclonal antibody (red, panel B and C) and ACSL6 protein was detected with a rabbit antibody (green, panel D). The inclusion membrane of one representative inclusion is indicated with an arrow in panel A and B. Panel C displays a cropped portion of the image shown in panel B. Insets in panel C and D display the orthogonal (z, x) and (z, y) views of the deconvolved Z stack merged images. Images were taken with a Zeiss LSM710 confocal microscope at 63x magnification. The bars in panels and insets represent 10 µm. Note that the two insets of panel C were slightly scaled up compare to the main image. Nuclei and inclusion are indicated with N and Inc, respectively. ACSL3 protein was detected inside the inclusion (panel B and C) and ACSL6 was not (panel D).

    Journal: PLoS ONE

    Article Title: Eukaryotic Protein Recruitment into the Chlamydia Inclusion: Implications for Survival and Growth

    doi: 10.1371/journal.pone.0036843

    Figure Lengend Snippet: ACSL3 but not ACSL6 is recruited into the Ct inclusion. HeLa cells were grown on coverslips and infected with Ct strain D at a MOI of 1 in cell culture medium without antibiotics and cycloheximide. Cells were fixed 36 hr post- infection. DNA was detected with Hoechst 33258 dye (blue). Bacteria were detected with a rabbit antibody against IncA, which was stained with an anti-rabbit antibody labeled with AlexaFluor®488 revealing the inclusion membrane (green, panels A to C) or with a mouse antibody against LPS, which was stained with Cy TM 3-conjugated anti-mouse antibody (red, panel D). Long-chain acyl-CoA synthetase 3, ACSL3, was detected with a mouse monoclonal antibody (red, panel B and C) and ACSL6 protein was detected with a rabbit antibody (green, panel D). The inclusion membrane of one representative inclusion is indicated with an arrow in panel A and B. Panel C displays a cropped portion of the image shown in panel B. Insets in panel C and D display the orthogonal (z, x) and (z, y) views of the deconvolved Z stack merged images. Images were taken with a Zeiss LSM710 confocal microscope at 63x magnification. The bars in panels and insets represent 10 µm. Note that the two insets of panel C were slightly scaled up compare to the main image. Nuclei and inclusion are indicated with N and Inc, respectively. ACSL3 protein was detected inside the inclusion (panel B and C) and ACSL6 was not (panel D).

    Article Snippet: Images were taken with a Zeiss LSM710 confocal microscope at 40x magnification using the same exposure settings for the un-treated and treated coverslips.

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

    The nuclear ZNF23 protein is mobilized into the lumen of the Ct inclusion. HeLa cells were grown on coverslips and infected in cell culture medium without antibiotics and cycloheximide. Cells were infected at a MOI of 1 with Ct strain L 2 (panel A to D) and with D (panel E) and were fixed 36 hr post-infection. DNA was detected with the Hoechst dye (blue). In cells infected with L2, the bacteria were detected with a mouse antibody against LPS (red, panel B and D) and the ZNF23 protein was detected with a rabbit antibody (green, panel C and D). Z stack images were deconvolved and single channel Surpass 3D view of a representative infected cell is shown in panel A to C. Cropped longitudinal view of the Surpass (x, y, z) view of the merged image is shown in panel D. In Panel E, the inclusion membrane was detected with a rabbit antibody against the bacterial IncA protein (green) and the ZNF23 protein was detected with a mouse antibody (red), and the merged image with Hoechst staining is shown. Images were taken with a Zeiss LSM710 confocal microscope at 63x magnification. The bars in the panels represent 10 µm. Nuclei and inclusion are indicated with N and Inc, respectively.

    Journal: PLoS ONE

    Article Title: Eukaryotic Protein Recruitment into the Chlamydia Inclusion: Implications for Survival and Growth

    doi: 10.1371/journal.pone.0036843

    Figure Lengend Snippet: The nuclear ZNF23 protein is mobilized into the lumen of the Ct inclusion. HeLa cells were grown on coverslips and infected in cell culture medium without antibiotics and cycloheximide. Cells were infected at a MOI of 1 with Ct strain L 2 (panel A to D) and with D (panel E) and were fixed 36 hr post-infection. DNA was detected with the Hoechst dye (blue). In cells infected with L2, the bacteria were detected with a mouse antibody against LPS (red, panel B and D) and the ZNF23 protein was detected with a rabbit antibody (green, panel C and D). Z stack images were deconvolved and single channel Surpass 3D view of a representative infected cell is shown in panel A to C. Cropped longitudinal view of the Surpass (x, y, z) view of the merged image is shown in panel D. In Panel E, the inclusion membrane was detected with a rabbit antibody against the bacterial IncA protein (green) and the ZNF23 protein was detected with a mouse antibody (red), and the merged image with Hoechst staining is shown. Images were taken with a Zeiss LSM710 confocal microscope at 63x magnification. The bars in the panels represent 10 µm. Nuclei and inclusion are indicated with N and Inc, respectively.

    Article Snippet: Images were taken with a Zeiss LSM710 confocal microscope at 40x magnification using the same exposure settings for the un-treated and treated coverslips.

    Techniques: Infection, Cell Culture, Staining, Microscopy

    LPCAT1 enzyme is tightly associated with the inclusion membrane. HeLa cells were grown on coverslips in cell culture medium without antibiotics and cycloheximide, and were transfected with a DNA construct expressing a GFP protein fused to acyl-CoA:lysophosphatidylcholine acyltransferase 1, LPCAT1. After 24 hr, cells were infected with Ct strain D at a MOI of 1 and were fixed 44 hr post-infection. DNA was detected with the Hoechst dye (blue) and the bacteria were detected with a rabbit antibody against IncA revealing the inclusion membrane (red). A deconvolved Z stack merged image with the GFP signal is shown in panel A. A cropped section of the image is displayed in the inset. White arrowheads indicate the position of the inclusion membrane. The tight association of LPCAT1with the inclusion membrane and overlap of the IncA and GFP signals, detected as yellow dots, can be observed in the inset. Panel B shows traces of the intensity of the signal for DNA, IncA and LPCAT1 (y axis) plotted in function of the distance (x axis) from area I to II as indicated by a red arrow on panel A. GFP and IncA signals co-localized in area I but not in area II. Images were taken with a Zeiss LSM710 confocal microscope at 63x magnification. The bars in the panel and the inset represent 10 and 1 µm, respectively.

    Journal: PLoS ONE

    Article Title: Eukaryotic Protein Recruitment into the Chlamydia Inclusion: Implications for Survival and Growth

    doi: 10.1371/journal.pone.0036843

    Figure Lengend Snippet: LPCAT1 enzyme is tightly associated with the inclusion membrane. HeLa cells were grown on coverslips in cell culture medium without antibiotics and cycloheximide, and were transfected with a DNA construct expressing a GFP protein fused to acyl-CoA:lysophosphatidylcholine acyltransferase 1, LPCAT1. After 24 hr, cells were infected with Ct strain D at a MOI of 1 and were fixed 44 hr post-infection. DNA was detected with the Hoechst dye (blue) and the bacteria were detected with a rabbit antibody against IncA revealing the inclusion membrane (red). A deconvolved Z stack merged image with the GFP signal is shown in panel A. A cropped section of the image is displayed in the inset. White arrowheads indicate the position of the inclusion membrane. The tight association of LPCAT1with the inclusion membrane and overlap of the IncA and GFP signals, detected as yellow dots, can be observed in the inset. Panel B shows traces of the intensity of the signal for DNA, IncA and LPCAT1 (y axis) plotted in function of the distance (x axis) from area I to II as indicated by a red arrow on panel A. GFP and IncA signals co-localized in area I but not in area II. Images were taken with a Zeiss LSM710 confocal microscope at 63x magnification. The bars in the panel and the inset represent 10 and 1 µm, respectively.

    Article Snippet: Images were taken with a Zeiss LSM710 confocal microscope at 40x magnification using the same exposure settings for the un-treated and treated coverslips.

    Techniques: Cell Culture, Transfection, Construct, Expressing, Infection, Microscopy

    Redistribution of ACBD6 from the nucleus and cytosol into the inclusion lumen during Ct development. HeLa cells were grown on coverslips and infected with Ct strain D at a MOI of 1 in cell culture medium without antibiotics and cycloheximide. After infection, cells were fixed at the indicated time (panel A to D). ACBD6 (green), Ct LPS (red) and DNA (Hoechst dye, blue) were detected as described in legend of Figure 3 . For each time point, the staining obtained for ACDB6 and the merge image obtained for ACBD6, LPS and DNA are shown on the left and right on each panel, respectively. Deconvolved Z stack images were analyzed with the Imaris Software package. Panel E shows the longitudinal Surpass view (x, y, z) of the distribution of ACBD6, LPS and DNA in one infected-cell 24 hr post-infection. Note that ACBD6 was detected inside the same space occupied by the bacteria in the inclusion, but did not co-localize with the LPS signal. Images were taken with a Zeiss LSM710 confocal microscope at 63x magnification. The bars in panels represent 10 µm. Representative inclusions are indicated with white arrows and nuclei by a capital N.

    Journal: PLoS ONE

    Article Title: Eukaryotic Protein Recruitment into the Chlamydia Inclusion: Implications for Survival and Growth

    doi: 10.1371/journal.pone.0036843

    Figure Lengend Snippet: Redistribution of ACBD6 from the nucleus and cytosol into the inclusion lumen during Ct development. HeLa cells were grown on coverslips and infected with Ct strain D at a MOI of 1 in cell culture medium without antibiotics and cycloheximide. After infection, cells were fixed at the indicated time (panel A to D). ACBD6 (green), Ct LPS (red) and DNA (Hoechst dye, blue) were detected as described in legend of Figure 3 . For each time point, the staining obtained for ACDB6 and the merge image obtained for ACBD6, LPS and DNA are shown on the left and right on each panel, respectively. Deconvolved Z stack images were analyzed with the Imaris Software package. Panel E shows the longitudinal Surpass view (x, y, z) of the distribution of ACBD6, LPS and DNA in one infected-cell 24 hr post-infection. Note that ACBD6 was detected inside the same space occupied by the bacteria in the inclusion, but did not co-localize with the LPS signal. Images were taken with a Zeiss LSM710 confocal microscope at 63x magnification. The bars in panels represent 10 µm. Representative inclusions are indicated with white arrows and nuclei by a capital N.

    Article Snippet: Images were taken with a Zeiss LSM710 confocal microscope at 40x magnification using the same exposure settings for the un-treated and treated coverslips.

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

    The N-terminal 63 amino acids of UL31 are sufficient to recruit EGFP to sites of laser microirradiation. Images of a live HeLa cell transfected with EGFP-N63 immediately before (Pre), immediately after (Post), and 30 seconds were acquired after the area indicated by the arrowhead was irradiated using an Olympus FV1000 laser-scanning confocal microscope. Images shown are representative of n = 12 cells analyzed in two independent experiments. Error bars are standard error of the mean (n = 12).

    Journal: Scientific Reports

    Article Title: The Herpesvirus Nuclear Egress Complex Component, UL31, Can Be Recruited to Sites of DNA Damage Through Poly-ADP Ribose Binding

    doi: 10.1038/s41598-017-02109-0

    Figure Lengend Snippet: The N-terminal 63 amino acids of UL31 are sufficient to recruit EGFP to sites of laser microirradiation. Images of a live HeLa cell transfected with EGFP-N63 immediately before (Pre), immediately after (Post), and 30 seconds were acquired after the area indicated by the arrowhead was irradiated using an Olympus FV1000 laser-scanning confocal microscope. Images shown are representative of n = 12 cells analyzed in two independent experiments. Error bars are standard error of the mean (n = 12).

    Article Snippet: Determination of nuclear:cytoplasmic ratio and Δmax Using an Olympus FV1000 confocal microscope and Fluoview software version 1.7.3.0, the fluorescence intensity of defined regions within the nuclei and cytoplasm of transfected cells were measured and used to calculate nucleus to cytoplasm ratios (N/C).

    Techniques: Transfection, Irradiation, Microscopy

    Recruitment of UL31 to sites of laser microirradiation requires its N-terminal 45 amino acids but not CR1. ( A ) Cartoon of HSV-2 UL31 identifying key features of the molecule including the position of the bipartite nuclear localization signal (NLS) as well as the positions of conserved regions (CR) 1 through 4. ( B ) Images of live HeLa cells expressing wild type EGFP-UL31 (WT), EGFP-UL31 lacking the CR1 region required for UL34 binding (ΔCR1), or EGFP-UL31 lacking its N-terminal 45 amino acids (Δ45) acquired immediately before (Pre), immediately after (Post), and 30 seconds after the areas indicated by arrowheads were microirradiated using an Olympus FV1000 laser-scanning confocal microscope. The graphs display quantitation of the average fluorescence intensity of the irradiated area (Bleach), as well as distal control areas (Control), over time. Images shown are representative of n = 12 cells analyzed in two independent experiments. Error bars are standard error of the mean (n = 12 per condition).

    Journal: Scientific Reports

    Article Title: The Herpesvirus Nuclear Egress Complex Component, UL31, Can Be Recruited to Sites of DNA Damage Through Poly-ADP Ribose Binding

    doi: 10.1038/s41598-017-02109-0

    Figure Lengend Snippet: Recruitment of UL31 to sites of laser microirradiation requires its N-terminal 45 amino acids but not CR1. ( A ) Cartoon of HSV-2 UL31 identifying key features of the molecule including the position of the bipartite nuclear localization signal (NLS) as well as the positions of conserved regions (CR) 1 through 4. ( B ) Images of live HeLa cells expressing wild type EGFP-UL31 (WT), EGFP-UL31 lacking the CR1 region required for UL34 binding (ΔCR1), or EGFP-UL31 lacking its N-terminal 45 amino acids (Δ45) acquired immediately before (Pre), immediately after (Post), and 30 seconds after the areas indicated by arrowheads were microirradiated using an Olympus FV1000 laser-scanning confocal microscope. The graphs display quantitation of the average fluorescence intensity of the irradiated area (Bleach), as well as distal control areas (Control), over time. Images shown are representative of n = 12 cells analyzed in two independent experiments. Error bars are standard error of the mean (n = 12 per condition).

    Article Snippet: Determination of nuclear:cytoplasmic ratio and Δmax Using an Olympus FV1000 confocal microscope and Fluoview software version 1.7.3.0, the fluorescence intensity of defined regions within the nuclei and cytoplasm of transfected cells were measured and used to calculate nucleus to cytoplasm ratios (N/C).

    Techniques: Expressing, Binding Assay, Microscopy, Quantitation Assay, Fluorescence, Irradiation

    UL31 recruitment to sites of laser microirradiation is not prevented by ATM inhibition ( A ) HeLa cells on grid-bottom dishes expressing EGFP-UL31 were treated with 0.1% DMSO (vehicle) or 10 μM KU-55933, an ATM inhibitor, two hours prior to imaging. Images of cells were captured immediately before (Pre), and immediately after (Post) the areas indicated by the white circles were microirradiated using an Olympus FV1000 laser-scanning confocal microscope. Cells were immediately fixed and stained with anti-γH2AX antibody followed by an Alexa 568 conjugated secondary antibody (red signal). Microirradiated cells were relocated and images captured by confocal microscopy. Representative images of 4 cells/condition in two independent experiments are shown. ( B ) Δmax of DMSO and KU-55933 treated cells was determined as described above (n = 8 cells/condition). Error bars represent standard error of the mean. Using an unpaired t -test the Δmax of DMSO treated cells was compared to KU-55933 treated cells and found not to be significant (ns).

    Journal: Scientific Reports

    Article Title: The Herpesvirus Nuclear Egress Complex Component, UL31, Can Be Recruited to Sites of DNA Damage Through Poly-ADP Ribose Binding

    doi: 10.1038/s41598-017-02109-0

    Figure Lengend Snippet: UL31 recruitment to sites of laser microirradiation is not prevented by ATM inhibition ( A ) HeLa cells on grid-bottom dishes expressing EGFP-UL31 were treated with 0.1% DMSO (vehicle) or 10 μM KU-55933, an ATM inhibitor, two hours prior to imaging. Images of cells were captured immediately before (Pre), and immediately after (Post) the areas indicated by the white circles were microirradiated using an Olympus FV1000 laser-scanning confocal microscope. Cells were immediately fixed and stained with anti-γH2AX antibody followed by an Alexa 568 conjugated secondary antibody (red signal). Microirradiated cells were relocated and images captured by confocal microscopy. Representative images of 4 cells/condition in two independent experiments are shown. ( B ) Δmax of DMSO and KU-55933 treated cells was determined as described above (n = 8 cells/condition). Error bars represent standard error of the mean. Using an unpaired t -test the Δmax of DMSO treated cells was compared to KU-55933 treated cells and found not to be significant (ns).

    Article Snippet: Determination of nuclear:cytoplasmic ratio and Δmax Using an Olympus FV1000 confocal microscope and Fluoview software version 1.7.3.0, the fluorescence intensity of defined regions within the nuclei and cytoplasm of transfected cells were measured and used to calculate nucleus to cytoplasm ratios (N/C).

    Techniques: Inhibition, Expressing, Imaging, Microscopy, Staining, Confocal Microscopy

    Identification of regions of UL31 required for nuclear localization and recruitment to sites of laser microirradiation. ( A ) Sequence of the N-terminal 63 amino acids of HSV-2 UL31. Serine (S) and threonine (T) residues highlighted in red are predicted to be modified by Us3 16 . Underlined residues are 75% similar to a canonical poly-ADP-ribose binding motif. Various C-terminal and N-terminal truncations of UL31 analyzed in these experiments are shown. ( B ) Images of live HeLa cells transfected with plasmids expressing the indicated EGFP tagged protein were captured using an Olympus FV1000 laser-scanning confocal microscope over the course of two independent experiments with n ≥ 7 cells/experiment. The relative fluorescence intensity in the nucleus and the cytoplasm was determined using Fluoview software version 1.7.3.0 and the nucleus:cytoplasm ratio calculated. Error bars represent standard error of the mean. Using an unpaired t test the nucleus to cytoplasm ratio of WT UL31 was compared to the other proteins (ns = not significant, *p

    Journal: Scientific Reports

    Article Title: The Herpesvirus Nuclear Egress Complex Component, UL31, Can Be Recruited to Sites of DNA Damage Through Poly-ADP Ribose Binding

    doi: 10.1038/s41598-017-02109-0

    Figure Lengend Snippet: Identification of regions of UL31 required for nuclear localization and recruitment to sites of laser microirradiation. ( A ) Sequence of the N-terminal 63 amino acids of HSV-2 UL31. Serine (S) and threonine (T) residues highlighted in red are predicted to be modified by Us3 16 . Underlined residues are 75% similar to a canonical poly-ADP-ribose binding motif. Various C-terminal and N-terminal truncations of UL31 analyzed in these experiments are shown. ( B ) Images of live HeLa cells transfected with plasmids expressing the indicated EGFP tagged protein were captured using an Olympus FV1000 laser-scanning confocal microscope over the course of two independent experiments with n ≥ 7 cells/experiment. The relative fluorescence intensity in the nucleus and the cytoplasm was determined using Fluoview software version 1.7.3.0 and the nucleus:cytoplasm ratio calculated. Error bars represent standard error of the mean. Using an unpaired t test the nucleus to cytoplasm ratio of WT UL31 was compared to the other proteins (ns = not significant, *p

    Article Snippet: Determination of nuclear:cytoplasmic ratio and Δmax Using an Olympus FV1000 confocal microscope and Fluoview software version 1.7.3.0, the fluorescence intensity of defined regions within the nuclei and cytoplasm of transfected cells were measured and used to calculate nucleus to cytoplasm ratios (N/C).

    Techniques: Sequencing, Modification, Binding Assay, Transfection, Expressing, Microscopy, Fluorescence, Software

    UL31 accumulates at sites of laser microirradiation-induced DNA-damage. ( A ) Representative images of live HeLa cells transfected with EGFP or EGFP-UL31 expression plasmids immediately before (Pre), immediately after (Post), and 30 seconds after the areas indicated by the arrowheads were microirradiated with a 405 nm laser using an Olympus FV1000 laser-scanning confocal microscope. The graphs display quantitation of the average fluorescence intensity of the microirradiated area (Bleach), as well as control distal areas (Control), over time for n = 20 cells in four independent experiments for EGFP-UL31 and for n = 22 cells in three independent experiments for EGFP. Error bars are standard error of the mean. ( B ) Images of a live HeLa cell transfected with EGFP-UL31 on grid-bottom dishes immediately before, and immediately after the indicated area (white circle) was microirradiated with a 405 nm laser using an Olympus FV1000 laser-scanning confocal microscope (Live). Cells were immediately fixed and stained with anti-γH2AX antibody followed by staining with Alexa 568 conjugated donkey anti-mouse secondary antibody. Microirradiated cells were relocated and images captured by confocal microscopy (Fixed). Image is representative of n = 8 cells analyzed in two independent experiments.

    Journal: Scientific Reports

    Article Title: The Herpesvirus Nuclear Egress Complex Component, UL31, Can Be Recruited to Sites of DNA Damage Through Poly-ADP Ribose Binding

    doi: 10.1038/s41598-017-02109-0

    Figure Lengend Snippet: UL31 accumulates at sites of laser microirradiation-induced DNA-damage. ( A ) Representative images of live HeLa cells transfected with EGFP or EGFP-UL31 expression plasmids immediately before (Pre), immediately after (Post), and 30 seconds after the areas indicated by the arrowheads were microirradiated with a 405 nm laser using an Olympus FV1000 laser-scanning confocal microscope. The graphs display quantitation of the average fluorescence intensity of the microirradiated area (Bleach), as well as control distal areas (Control), over time for n = 20 cells in four independent experiments for EGFP-UL31 and for n = 22 cells in three independent experiments for EGFP. Error bars are standard error of the mean. ( B ) Images of a live HeLa cell transfected with EGFP-UL31 on grid-bottom dishes immediately before, and immediately after the indicated area (white circle) was microirradiated with a 405 nm laser using an Olympus FV1000 laser-scanning confocal microscope (Live). Cells were immediately fixed and stained with anti-γH2AX antibody followed by staining with Alexa 568 conjugated donkey anti-mouse secondary antibody. Microirradiated cells were relocated and images captured by confocal microscopy (Fixed). Image is representative of n = 8 cells analyzed in two independent experiments.

    Article Snippet: Determination of nuclear:cytoplasmic ratio and Δmax Using an Olympus FV1000 confocal microscope and Fluoview software version 1.7.3.0, the fluorescence intensity of defined regions within the nuclei and cytoplasm of transfected cells were measured and used to calculate nucleus to cytoplasm ratios (N/C).

    Techniques: Transfection, Expressing, Microscopy, Quantitation Assay, Fluorescence, Staining, Confocal Microscopy

    ( a ) Serum-deprived cells present a quiescent phenotype to manage oxidative stress. (i) One hundred fifty thousand (1.5 × 10 5 ) PC3 prostate cancer cells were plated with or without serum (24 h) prior to treatment with 250 μM H 2 0 2 for 4 h. Phase contrast microscopy (20×) was captured cell morphology. (ii) One million (1 × 10 6 ) serum-containing and serum-deprived PC3 prostate cancer cells were pre-treated with 5 mM n-acetyl-cysteine (NAC) for 1 h prior to treatment with 250 μM H 2 O 2 . Quiescence was determined via Western blot analysis for phosphorylated-retinoblastoma (pRB) or p27 Kip1 protein expression. β-actin served as a loading control. Pictures of gels/blots were cropped to focus on target protein expression. Full length gels/blots are included in supplementary figure 1 . (iii) One hundred fifty thousand (1.5 × 10 5 ) cells were treated as mentioned above and fixed in 4% paraformaldehyde prior to incubation with rabbit anti-p27 Kip1 and mouse anti-PMCA antibodies, followed by Cy3-conjugated anti-rabbit and Alexa Fluor-488 anti-mouse antibodies. Imaging of cells (63×) was performed on a Zeiss LSM700 Confocal Microscope. Scale bar = 50 μm. ( b ) Serum-deprived cells present a quiescent phenotype to manage oxidative stress. (i) One million (1.0 × 10 6 ) DU145 prostate cancer cells were plated with or without serum (24 h) prior to treatment with 250 μM H 2 0 2 for 4 h. Phase contrast microscopy (20×) was utilized to captured cell morphology. (ii) One million (1 × 10 6 ) serum-containing and serum-deprived DU145 prostate cancer cells were pre-treated with 5 mM n-acetyl-cysteine (NAC) for 1 h prior to treatment with 250 μM H 2 O 2 . Quiescence was determined via Western blot analysis for phosphorylated-retinoblastoma (pRB) or p27 Kip1 protein expression. β-actin served as a loading control. Pictures of gels/blots were cropped to focus on target protein expression. Full length gels/blots are included in supplementary figure 2 . (iii) One hundred fifty thousand (1.5 × 10 5 ) DU145 prostate cancer cells were treated as mentioned above and fixed in 4% paraformaldehyde prior to incubation with rabbit anti-p27 Kip1 and mouse anti-PMCA antibodies, followed by Cy3-conjugated anti-rabbit and Alexa Fluor-488 anti-mouse antibodies. Imaging of cells (63×) was performed on a Zeiss LSM-700 Confocal Microscope. Scale bar = 50 μm.

    Journal: Scientific Reports

    Article Title: Serum deprivation initiates adaptation and survival to oxidative stress in prostate cancer cells

    doi: 10.1038/s41598-020-68668-x

    Figure Lengend Snippet: ( a ) Serum-deprived cells present a quiescent phenotype to manage oxidative stress. (i) One hundred fifty thousand (1.5 × 10 5 ) PC3 prostate cancer cells were plated with or without serum (24 h) prior to treatment with 250 μM H 2 0 2 for 4 h. Phase contrast microscopy (20×) was captured cell morphology. (ii) One million (1 × 10 6 ) serum-containing and serum-deprived PC3 prostate cancer cells were pre-treated with 5 mM n-acetyl-cysteine (NAC) for 1 h prior to treatment with 250 μM H 2 O 2 . Quiescence was determined via Western blot analysis for phosphorylated-retinoblastoma (pRB) or p27 Kip1 protein expression. β-actin served as a loading control. Pictures of gels/blots were cropped to focus on target protein expression. Full length gels/blots are included in supplementary figure 1 . (iii) One hundred fifty thousand (1.5 × 10 5 ) cells were treated as mentioned above and fixed in 4% paraformaldehyde prior to incubation with rabbit anti-p27 Kip1 and mouse anti-PMCA antibodies, followed by Cy3-conjugated anti-rabbit and Alexa Fluor-488 anti-mouse antibodies. Imaging of cells (63×) was performed on a Zeiss LSM700 Confocal Microscope. Scale bar = 50 μm. ( b ) Serum-deprived cells present a quiescent phenotype to manage oxidative stress. (i) One million (1.0 × 10 6 ) DU145 prostate cancer cells were plated with or without serum (24 h) prior to treatment with 250 μM H 2 0 2 for 4 h. Phase contrast microscopy (20×) was utilized to captured cell morphology. (ii) One million (1 × 10 6 ) serum-containing and serum-deprived DU145 prostate cancer cells were pre-treated with 5 mM n-acetyl-cysteine (NAC) for 1 h prior to treatment with 250 μM H 2 O 2 . Quiescence was determined via Western blot analysis for phosphorylated-retinoblastoma (pRB) or p27 Kip1 protein expression. β-actin served as a loading control. Pictures of gels/blots were cropped to focus on target protein expression. Full length gels/blots are included in supplementary figure 2 . (iii) One hundred fifty thousand (1.5 × 10 5 ) DU145 prostate cancer cells were treated as mentioned above and fixed in 4% paraformaldehyde prior to incubation with rabbit anti-p27 Kip1 and mouse anti-PMCA antibodies, followed by Cy3-conjugated anti-rabbit and Alexa Fluor-488 anti-mouse antibodies. Imaging of cells (63×) was performed on a Zeiss LSM-700 Confocal Microscope. Scale bar = 50 μm.

    Article Snippet: Images were taken with Zeiss LSM-700 Confocal Microscope at excitation 488 nm for Alexa Fluor and 550 nm for Cy3.

    Techniques: Microscopy, Western Blot, Expressing, Incubation, Imaging

    Mirk/Dyrk1B inhibitors, AZ191 and NCGC00185981-05/ML195, inhibit oxidative stress-induced quiescence. One hundred thousand (1 × 10 5 ) DU145 cells were harvested for immunocytochemistry as described above prior to stimulating different concentrations (1, 2, 3, 5, and 10 µM) of Mirk/DYRK1B inhibitors: ( a ) AZ191 and ( b ) NCGC00185981-05/ML195 prior to culturing in serum-free media (24 h) stimulated with 250 μM H 2 O 2 for 1 h to induce quiescence. Cells were then fixed with 4% paraformaldehyde, blocked, then incubated with an antibody mixture containing a rabbit p27 Kip1 antibody and mouse anti-PMCA antibody, followed by secondary mixture containing a Cy3-conjugated anti-rabbit antibody and Alexa Fluor-488 anti-mouse antibody. DMSO served as a control. Imaging of cells (63×) was with a Zeiss LSM-700 Confocal Microscope. Scale bar = 50 μm.

    Journal: Scientific Reports

    Article Title: Serum deprivation initiates adaptation and survival to oxidative stress in prostate cancer cells

    doi: 10.1038/s41598-020-68668-x

    Figure Lengend Snippet: Mirk/Dyrk1B inhibitors, AZ191 and NCGC00185981-05/ML195, inhibit oxidative stress-induced quiescence. One hundred thousand (1 × 10 5 ) DU145 cells were harvested for immunocytochemistry as described above prior to stimulating different concentrations (1, 2, 3, 5, and 10 µM) of Mirk/DYRK1B inhibitors: ( a ) AZ191 and ( b ) NCGC00185981-05/ML195 prior to culturing in serum-free media (24 h) stimulated with 250 μM H 2 O 2 for 1 h to induce quiescence. Cells were then fixed with 4% paraformaldehyde, blocked, then incubated with an antibody mixture containing a rabbit p27 Kip1 antibody and mouse anti-PMCA antibody, followed by secondary mixture containing a Cy3-conjugated anti-rabbit antibody and Alexa Fluor-488 anti-mouse antibody. DMSO served as a control. Imaging of cells (63×) was with a Zeiss LSM-700 Confocal Microscope. Scale bar = 50 μm.

    Article Snippet: Images were taken with Zeiss LSM-700 Confocal Microscope at excitation 488 nm for Alexa Fluor and 550 nm for Cy3.

    Techniques: Immunocytochemistry, Incubation, Imaging, Microscopy

    Quiescence inhibitors prevented nuclear localization of RelA/p65 (NF-κB). One hundred thousand (1 × 10 5 ) DU145 prostate cancer cells were harvested for immunocytochemistry as described above plated on glass coverslips in 6-well plates. Cells were harvested with different concentrations (1, 2, 3, 5, and 10 µM) of AZ191. Cells were stimulated with different concentrations of AZ191 (3, 5, and 10 µM) of two different prior to culturing in serum-free media (24 h) stimulated with 250 μM H 2 O 2 for 1 h to induce quiescence. Cells were then fixed with 4% paraformaldehyde, blocked, then incubated with an antibody mixture containing a rabbit NF-κB antibody and mouse anti-PMCA antibody, followed by secondary mixture containing a Cy3-conjugated anti-rabbit antibody and Alexa Fluor-488 anti-mouse antibody. DMSO was utilized as a control. Imaging of cells (63×) was with a Zeiss LSM-700 Confocal Microscope. Scale bar = 50 μm.

    Journal: Scientific Reports

    Article Title: Serum deprivation initiates adaptation and survival to oxidative stress in prostate cancer cells

    doi: 10.1038/s41598-020-68668-x

    Figure Lengend Snippet: Quiescence inhibitors prevented nuclear localization of RelA/p65 (NF-κB). One hundred thousand (1 × 10 5 ) DU145 prostate cancer cells were harvested for immunocytochemistry as described above plated on glass coverslips in 6-well plates. Cells were harvested with different concentrations (1, 2, 3, 5, and 10 µM) of AZ191. Cells were stimulated with different concentrations of AZ191 (3, 5, and 10 µM) of two different prior to culturing in serum-free media (24 h) stimulated with 250 μM H 2 O 2 for 1 h to induce quiescence. Cells were then fixed with 4% paraformaldehyde, blocked, then incubated with an antibody mixture containing a rabbit NF-κB antibody and mouse anti-PMCA antibody, followed by secondary mixture containing a Cy3-conjugated anti-rabbit antibody and Alexa Fluor-488 anti-mouse antibody. DMSO was utilized as a control. Imaging of cells (63×) was with a Zeiss LSM-700 Confocal Microscope. Scale bar = 50 μm.

    Article Snippet: Images were taken with Zeiss LSM-700 Confocal Microscope at excitation 488 nm for Alexa Fluor and 550 nm for Cy3.

    Techniques: Immunocytochemistry, Incubation, Imaging, Microscopy

    RelA/p65 (NF-κB) translocates to the nucleus in response to oxidative stress. ( a , b ) Two million (2 × 10 6 ) PC3 and DU145 cells were harvested with or without serum for 24 h prior to pre-treatment with 5 mM n-acetyl-cysteine (NAC) for 1 h followed by 250 μM H 2 O 2 for 1 h, or TNFα (0.1 ng/mL; 30 min). Proteins were harvested via subcellular fractionation according to the manufacturer’s instruction and resolved by Western blot analysis. Immunoblots were probed with anti-RelA/p65; anti-β-actin (non-nuclear) and anti-Topoisomerase1 (Topo 1, nuclear) served as markers for fractionation purity and as loading controls. Pictures of gel/blots were cropped to focus on target protein expression. Full length gels/blots are included in supplementary figure 3 (PC3) and 4 (DU145). ( c , d ) One hundred thousand (1 × 10 5 ) PC3 and DU145 cells treated as mentioned above and were harvested for immunocytochemistry, fixed with 4% paraformaldehyde, blocked, then incubated with rabbit anti-RelA/p65 and mouse anti-PMCA antibodies, followed by Cy3-conjugated anti-rabbit and FITC-conjugated anti-mouse antibodies. Imaging of cells (63×) was performed on a Zeiss LSM-700 Confocal Microscope. Scale bar = 50 μm.

    Journal: Scientific Reports

    Article Title: Serum deprivation initiates adaptation and survival to oxidative stress in prostate cancer cells

    doi: 10.1038/s41598-020-68668-x

    Figure Lengend Snippet: RelA/p65 (NF-κB) translocates to the nucleus in response to oxidative stress. ( a , b ) Two million (2 × 10 6 ) PC3 and DU145 cells were harvested with or without serum for 24 h prior to pre-treatment with 5 mM n-acetyl-cysteine (NAC) for 1 h followed by 250 μM H 2 O 2 for 1 h, or TNFα (0.1 ng/mL; 30 min). Proteins were harvested via subcellular fractionation according to the manufacturer’s instruction and resolved by Western blot analysis. Immunoblots were probed with anti-RelA/p65; anti-β-actin (non-nuclear) and anti-Topoisomerase1 (Topo 1, nuclear) served as markers for fractionation purity and as loading controls. Pictures of gel/blots were cropped to focus on target protein expression. Full length gels/blots are included in supplementary figure 3 (PC3) and 4 (DU145). ( c , d ) One hundred thousand (1 × 10 5 ) PC3 and DU145 cells treated as mentioned above and were harvested for immunocytochemistry, fixed with 4% paraformaldehyde, blocked, then incubated with rabbit anti-RelA/p65 and mouse anti-PMCA antibodies, followed by Cy3-conjugated anti-rabbit and FITC-conjugated anti-mouse antibodies. Imaging of cells (63×) was performed on a Zeiss LSM-700 Confocal Microscope. Scale bar = 50 μm.

    Article Snippet: Images were taken with Zeiss LSM-700 Confocal Microscope at excitation 488 nm for Alexa Fluor and 550 nm for Cy3.

    Techniques: Fractionation, Western Blot, Expressing, Immunocytochemistry, Incubation, Imaging, Microscopy

    At late times postinfection with SIN/nsP3GFP and ns polyprotein cleavage mutants, SINV-specific dsRNAs retain mostly plasma membrane-specific localization. BHK-21 cells were infected with the indicated viruses at an MOI of 20 PFU/cell. At 8 h (and 16 h) postinfection, cells were fixed, permeabilized, and stained with a dsRNA-specific MAb. Images were acquired on a Zeiss LSM700 confocal microscope with a 63× 1.4NA PlanApochromat oil objective. The image stacks were further processed using Huygens Professional deconvolution and Imaris 3D rendering software. The xy images are presented as multiple-intensity projections of the entire stack, and xz sections are presented as a multiple-intensity projection of 2-μm section. Bars correspond to 5 μm. White arrowheads indicate positions of stained dsRNAs demonstrating cytoplasmic localization.

    Journal: Journal of Virology

    Article Title: Functional Sindbis Virus Replicative Complexes Are Formed at the Plasma Membrane ▿

    doi: 10.1128/JVI.01441-10

    Figure Lengend Snippet: At late times postinfection with SIN/nsP3GFP and ns polyprotein cleavage mutants, SINV-specific dsRNAs retain mostly plasma membrane-specific localization. BHK-21 cells were infected with the indicated viruses at an MOI of 20 PFU/cell. At 8 h (and 16 h) postinfection, cells were fixed, permeabilized, and stained with a dsRNA-specific MAb. Images were acquired on a Zeiss LSM700 confocal microscope with a 63× 1.4NA PlanApochromat oil objective. The image stacks were further processed using Huygens Professional deconvolution and Imaris 3D rendering software. The xy images are presented as multiple-intensity projections of the entire stack, and xz sections are presented as a multiple-intensity projection of 2-μm section. Bars correspond to 5 μm. White arrowheads indicate positions of stained dsRNAs demonstrating cytoplasmic localization.

    Article Snippet: Images were acquired on a Zeiss LSM700 confocal microscope with a 63× 1.4NA PlanApochromat oil objective.

    Techniques: Infection, Staining, Microscopy, Software

    At 2 h postinfection with SIN/nsP3GFP and ns polyprotein cleavage mutants, virus-specific dsRNAs are located at the plasma membrane. BHK-21 cells were infected with the indicated viruses at an MOI of 20 PFU/cell. At 2 h postinfection, they were fixed, permeabilized, and stained with dsRNA-specific MAb. The 3D image stacks were acquired on a Zeiss LSM700 confocal microscope with a 63× 1.4NA PlanApochromat oil objective. They were further processed using Huygens Professional deconvolution and Imaris 3D rendering software. The xy images are presented as multiple-intensity projections of the entire stack, and xz sections are presented as multiple-intensity projections of 2-μm section. Bars correspond to 5 μm.

    Journal: Journal of Virology

    Article Title: Functional Sindbis Virus Replicative Complexes Are Formed at the Plasma Membrane ▿

    doi: 10.1128/JVI.01441-10

    Figure Lengend Snippet: At 2 h postinfection with SIN/nsP3GFP and ns polyprotein cleavage mutants, virus-specific dsRNAs are located at the plasma membrane. BHK-21 cells were infected with the indicated viruses at an MOI of 20 PFU/cell. At 2 h postinfection, they were fixed, permeabilized, and stained with dsRNA-specific MAb. The 3D image stacks were acquired on a Zeiss LSM700 confocal microscope with a 63× 1.4NA PlanApochromat oil objective. They were further processed using Huygens Professional deconvolution and Imaris 3D rendering software. The xy images are presented as multiple-intensity projections of the entire stack, and xz sections are presented as multiple-intensity projections of 2-μm section. Bars correspond to 5 μm.

    Article Snippet: Images were acquired on a Zeiss LSM700 confocal microscope with a 63× 1.4NA PlanApochromat oil objective.

    Techniques: Infection, Staining, Microscopy, Software

    At early times postinfection, dsRNAs are located in membrane spherules presented on the external surface of the plasma membrane. (A) BHK-21 cells were infected with SINV/nsP3GFP at an MOI of 20 PFU/cell. At 2 h postinfection, cells were fixed with 4% PFA, permeabilized with either 0.02% or 0.5% saponin, treated with dsRNA-specific MAb and rabbit anti-SINV nsP1 Ab, and further stained with appropriate secondary Abs. Images were acquired on a Zeiss LSM700 META confocal microscope with a 63× 1.4NA PlanApochromat oil objective. The image stacks were further processed using Huygens Professional deconvolution and Imaris 3D rendering software. Bars correspond to 10 μm. (B) BHK-21 cells were infected with SINV/nsP3GFP at an MOI of 20 PFU/cell. At 3 h postinfection, cells were fixed with 4% PFA, permeabilized with 0.02% saponin, treated with dsRNA-specific MAb and gold-labeled secondary Abs, and further processed for EM. (C) The same cell sample as in panel B was processed for EM without permeabilization and immunostaining.

    Journal: Journal of Virology

    Article Title: Functional Sindbis Virus Replicative Complexes Are Formed at the Plasma Membrane ▿

    doi: 10.1128/JVI.01441-10

    Figure Lengend Snippet: At early times postinfection, dsRNAs are located in membrane spherules presented on the external surface of the plasma membrane. (A) BHK-21 cells were infected with SINV/nsP3GFP at an MOI of 20 PFU/cell. At 2 h postinfection, cells were fixed with 4% PFA, permeabilized with either 0.02% or 0.5% saponin, treated with dsRNA-specific MAb and rabbit anti-SINV nsP1 Ab, and further stained with appropriate secondary Abs. Images were acquired on a Zeiss LSM700 META confocal microscope with a 63× 1.4NA PlanApochromat oil objective. The image stacks were further processed using Huygens Professional deconvolution and Imaris 3D rendering software. Bars correspond to 10 μm. (B) BHK-21 cells were infected with SINV/nsP3GFP at an MOI of 20 PFU/cell. At 3 h postinfection, cells were fixed with 4% PFA, permeabilized with 0.02% saponin, treated with dsRNA-specific MAb and gold-labeled secondary Abs, and further processed for EM. (C) The same cell sample as in panel B was processed for EM without permeabilization and immunostaining.

    Article Snippet: Images were acquired on a Zeiss LSM700 confocal microscope with a 63× 1.4NA PlanApochromat oil objective.

    Techniques: Infection, Staining, Microscopy, Software, Labeling, Immunostaining

    In situ PLA demonstrates colocalization of dsRNA with SINV nsP1, nsP2, and nsP3 proteins. (A and B) BHK-21 cells were infected with SINV/nsP3GFP at an MOI of 20 PFU/cell. At 2 h postinfection, cells were fixed with 4% PFA, permeabilized with either 0.02% (A) or 0.5% (B) saponin, treated with dsRNA-specific MAb and rabbit anti-SINV nsP1 Ab, and further processed for in situ PLA. Red signals indicate colocalization of nsP1 and dsRNA. Images were acquired on a Zeiss LSM700 confocal microscope with a 63× 1.4NA PlanApochromat oil objective. The image stacks were further processed using Huygens Professional deconvolution and Imaris 3D rendering software. The xy images are presented as multiple-intensity projections of the entire stacks, and xz sections are presented as multiple-intensity projections of a 2-μm section. Bars correspond to 5 μm. (C) Numbers of PLA-positive foci detected in the cells by using dsRNA MAbs and nsP-specific, affinity-purified Abs. The nsP1-, nsP2-, or nsP3-specific Abs used and the concentrations of saponin are indicated. The dsRNA-positive foci in 3D images were quantitated using a Spot function in Imaris 3D rendering software. The P values were calculated using the Mann-Whitney test. Average values are indicated in red. (D) Results of PLA performed using dsRNA-specific MAb and nsP1-specific rabbit Ab with mock-infected cells.

    Journal: Journal of Virology

    Article Title: Functional Sindbis Virus Replicative Complexes Are Formed at the Plasma Membrane ▿

    doi: 10.1128/JVI.01441-10

    Figure Lengend Snippet: In situ PLA demonstrates colocalization of dsRNA with SINV nsP1, nsP2, and nsP3 proteins. (A and B) BHK-21 cells were infected with SINV/nsP3GFP at an MOI of 20 PFU/cell. At 2 h postinfection, cells were fixed with 4% PFA, permeabilized with either 0.02% (A) or 0.5% (B) saponin, treated with dsRNA-specific MAb and rabbit anti-SINV nsP1 Ab, and further processed for in situ PLA. Red signals indicate colocalization of nsP1 and dsRNA. Images were acquired on a Zeiss LSM700 confocal microscope with a 63× 1.4NA PlanApochromat oil objective. The image stacks were further processed using Huygens Professional deconvolution and Imaris 3D rendering software. The xy images are presented as multiple-intensity projections of the entire stacks, and xz sections are presented as multiple-intensity projections of a 2-μm section. Bars correspond to 5 μm. (C) Numbers of PLA-positive foci detected in the cells by using dsRNA MAbs and nsP-specific, affinity-purified Abs. The nsP1-, nsP2-, or nsP3-specific Abs used and the concentrations of saponin are indicated. The dsRNA-positive foci in 3D images were quantitated using a Spot function in Imaris 3D rendering software. The P values were calculated using the Mann-Whitney test. Average values are indicated in red. (D) Results of PLA performed using dsRNA-specific MAb and nsP1-specific rabbit Ab with mock-infected cells.

    Article Snippet: Images were acquired on a Zeiss LSM700 confocal microscope with a 63× 1.4NA PlanApochromat oil objective.

    Techniques: In Situ, Proximity Ligation Assay, Infection, Microscopy, Software, Affinity Purification, MANN-WHITNEY

    The plasma membrane is the site of viral RNA synthesis. BHK-21 cells were infected with SINV/nsP3GFP at an MOI of ca. 20 PFU/cell. At 4 h postinfection, they were treated with digitonin (1 μg/ml) in the presence of ActD, and an in vitro transcription reaction was performed as described in Materials and Methods. Cells were then stained with BrU-specific and dsRNA-specific antibodies. Images were acquired on a Zeiss LSM700 confocal microscope with a 63× 1.4NA PlanApochromat oil objective. The image stacks were further processed using Huygens Professional deconvolution and Imaris 3D rendering software. The xy images are presented as multiple-intensity projections of the entire stack, and xz and yz sections are presented as multiple-intensity projections of a 0.06-μm section. Bars correspond to 5 μm. White arrowheads indicate sites of colocalization of dsRNA and incorporated BrU. The white arrow points to one of the BrU-positive foci that are not associated with dsRNA staining. Mock-infected cells demonstrated no detectable staining under these experimental conditions; therefore, their images are not presented.

    Journal: Journal of Virology

    Article Title: Functional Sindbis Virus Replicative Complexes Are Formed at the Plasma Membrane ▿

    doi: 10.1128/JVI.01441-10

    Figure Lengend Snippet: The plasma membrane is the site of viral RNA synthesis. BHK-21 cells were infected with SINV/nsP3GFP at an MOI of ca. 20 PFU/cell. At 4 h postinfection, they were treated with digitonin (1 μg/ml) in the presence of ActD, and an in vitro transcription reaction was performed as described in Materials and Methods. Cells were then stained with BrU-specific and dsRNA-specific antibodies. Images were acquired on a Zeiss LSM700 confocal microscope with a 63× 1.4NA PlanApochromat oil objective. The image stacks were further processed using Huygens Professional deconvolution and Imaris 3D rendering software. The xy images are presented as multiple-intensity projections of the entire stack, and xz and yz sections are presented as multiple-intensity projections of a 0.06-μm section. Bars correspond to 5 μm. White arrowheads indicate sites of colocalization of dsRNA and incorporated BrU. The white arrow points to one of the BrU-positive foci that are not associated with dsRNA staining. Mock-infected cells demonstrated no detectable staining under these experimental conditions; therefore, their images are not presented.

    Article Snippet: Images were acquired on a Zeiss LSM700 confocal microscope with a 63× 1.4NA PlanApochromat oil objective.

    Techniques: Infection, In Vitro, Staining, Microscopy, Software

    At early times postinfection of mosquito cells, nsP3-GFP and dsRNAs are distributed at both the plasma membrane and endosomes. C 7 10 cells were infected with SINV/nsP3GFP at an MOI of 20 PFU/cell. After 4 h of incubation at 30°C, cells were fixed, permeabilized, and stained with dsRNA-specific MAb and Alexa Fluor 555-labeled secondary Ab as described in Materials and Methods. Images were acquired on a Zeiss LSM700 confocal microscope with a 63× 1.4NA PlanApochromat oil objective, and image stacks were further processed using Huygens Professional deconvolution and Imaris 3D rendering software. The xy images are presented as multiple-intensity projections of the entire stack, and xz sections are presented as multiple-intensity projections of a 2-μm section. White arrowheads indicate positions of stained dsRNAs having a cytoplasmic, endosome-like distribution. White arrows indicate plasma membrane-associated dsRNAs. Bars correspond to 5 μm.

    Journal: Journal of Virology

    Article Title: Functional Sindbis Virus Replicative Complexes Are Formed at the Plasma Membrane ▿

    doi: 10.1128/JVI.01441-10

    Figure Lengend Snippet: At early times postinfection of mosquito cells, nsP3-GFP and dsRNAs are distributed at both the plasma membrane and endosomes. C 7 10 cells were infected with SINV/nsP3GFP at an MOI of 20 PFU/cell. After 4 h of incubation at 30°C, cells were fixed, permeabilized, and stained with dsRNA-specific MAb and Alexa Fluor 555-labeled secondary Ab as described in Materials and Methods. Images were acquired on a Zeiss LSM700 confocal microscope with a 63× 1.4NA PlanApochromat oil objective, and image stacks were further processed using Huygens Professional deconvolution and Imaris 3D rendering software. The xy images are presented as multiple-intensity projections of the entire stack, and xz sections are presented as multiple-intensity projections of a 2-μm section. White arrowheads indicate positions of stained dsRNAs having a cytoplasmic, endosome-like distribution. White arrows indicate plasma membrane-associated dsRNAs. Bars correspond to 5 μm.

    Article Snippet: Images were acquired on a Zeiss LSM700 confocal microscope with a 63× 1.4NA PlanApochromat oil objective.

    Techniques: Infection, Incubation, Staining, Labeling, Microscopy, Software

    Re-localization of spherules from the plasma membrane into the cytoplasm. (A) Plasma membrane invaginations and vacuole formation in BHK-21 cells, infected with SINV/nsP3GFP, at 3 h postinfection. The bar corresponds to 200 nm. (B) CPV1 in BHK-21 cells, infected with SINV/nsP3GFP, at 6 h postinfection. The bar corresponds to 100 nm. (C and D) Distribution of nsP1, nsP3-GFP, and dsRNA in the cells at 2 and 8 h postinfection with SINV/nsP3GFP, respectively. BHK-21 cells were infected with SINV/nsP3GFP at an MOI of 20 PFU/cell. At the indicated times, cells were fixed, permeabilized, and stained with dsRNA- and nsP1-specific Abs. Panels a, xy panels represent multiple-intensity projections of a 1-μm xy section of the cell fragment, and xz and yz panels represent multiple-intensity projections of a 2-μm xy section as denoted on the xy panel. Panels b, c, and d, enlarged single xz section of the cell presented in panels a. Panels b show the distributions of nsP1 (red) and dsRNA (blue). A magenta color indicates colocalization of dsRNA and nsP1. Panels c show the distributions of nsP3 (green) and dsRNA (blue). A cyan color indicates colocalization of dsRNA and nsP3. Panels d demonstrate the distributions of nsP1 (red), nsP3 (green), and dsRNA (blue). A white color indicates colocalization of dsRNA and nonstructural proteins nsP1 and nsP3. The white arrowhead indicates the position of one of the endosomes stained with nsP1- and dsRNA-specific Ab and positive for the presence of nsP3-GFP. The red arrowhead indicates one of the nsP1-positive cytoplasmic complexes that lack nsP3-GFP and dsRNA. White arrows point to the cytoplasmic dsRNA-positive foci lacking nsP3-GFP and nsP1 association. Images were acquired on a Zeiss LSM700 confocal microscope with a 63× 1.4NA PlanApochromat oil objective. The image stacks were further processed using Huygens Professional deconvolution and Imaris 3D rendering software. Bars correspond to 5 μm.

    Journal: Journal of Virology

    Article Title: Functional Sindbis Virus Replicative Complexes Are Formed at the Plasma Membrane ▿

    doi: 10.1128/JVI.01441-10

    Figure Lengend Snippet: Re-localization of spherules from the plasma membrane into the cytoplasm. (A) Plasma membrane invaginations and vacuole formation in BHK-21 cells, infected with SINV/nsP3GFP, at 3 h postinfection. The bar corresponds to 200 nm. (B) CPV1 in BHK-21 cells, infected with SINV/nsP3GFP, at 6 h postinfection. The bar corresponds to 100 nm. (C and D) Distribution of nsP1, nsP3-GFP, and dsRNA in the cells at 2 and 8 h postinfection with SINV/nsP3GFP, respectively. BHK-21 cells were infected with SINV/nsP3GFP at an MOI of 20 PFU/cell. At the indicated times, cells were fixed, permeabilized, and stained with dsRNA- and nsP1-specific Abs. Panels a, xy panels represent multiple-intensity projections of a 1-μm xy section of the cell fragment, and xz and yz panels represent multiple-intensity projections of a 2-μm xy section as denoted on the xy panel. Panels b, c, and d, enlarged single xz section of the cell presented in panels a. Panels b show the distributions of nsP1 (red) and dsRNA (blue). A magenta color indicates colocalization of dsRNA and nsP1. Panels c show the distributions of nsP3 (green) and dsRNA (blue). A cyan color indicates colocalization of dsRNA and nsP3. Panels d demonstrate the distributions of nsP1 (red), nsP3 (green), and dsRNA (blue). A white color indicates colocalization of dsRNA and nonstructural proteins nsP1 and nsP3. The white arrowhead indicates the position of one of the endosomes stained with nsP1- and dsRNA-specific Ab and positive for the presence of nsP3-GFP. The red arrowhead indicates one of the nsP1-positive cytoplasmic complexes that lack nsP3-GFP and dsRNA. White arrows point to the cytoplasmic dsRNA-positive foci lacking nsP3-GFP and nsP1 association. Images were acquired on a Zeiss LSM700 confocal microscope with a 63× 1.4NA PlanApochromat oil objective. The image stacks were further processed using Huygens Professional deconvolution and Imaris 3D rendering software. Bars correspond to 5 μm.

    Article Snippet: Images were acquired on a Zeiss LSM700 confocal microscope with a 63× 1.4NA PlanApochromat oil objective.

    Techniques: Infection, Staining, Microscopy, Software

    Immunolocalization of both ataxin‐2 B and C in the fat body cells of third instar larvae. Immunolabeling using α‐dAtx2 B + C and anti‐rabbit IgG conjugated to Alexa 488 (green). Actin filaments were stained with phalloidin conjugated to Alexa 594 (red) and nuclei with 4′,6‐diamidino‐2‐phenylindole, fluorescent stain that binds strongly to A‐T rich regions in DNA (blue). The images were captured using the Zeiss LSM 780 confocal microscope.

    Journal: FEBS Open Bio

    Article Title: Drosophila ataxin‐2 gene encodes two differentially expressed isoforms and its function in larval fat body is crucial for development of peripheral tissues

    doi: 10.1002/2211-5463.12124

    Figure Lengend Snippet: Immunolocalization of both ataxin‐2 B and C in the fat body cells of third instar larvae. Immunolabeling using α‐dAtx2 B + C and anti‐rabbit IgG conjugated to Alexa 488 (green). Actin filaments were stained with phalloidin conjugated to Alexa 594 (red) and nuclei with 4′,6‐diamidino‐2‐phenylindole, fluorescent stain that binds strongly to A‐T rich regions in DNA (blue). The images were captured using the Zeiss LSM 780 confocal microscope.

    Article Snippet: The samples were examined using Leica TCS SP5 confocal microscope or Zeiss LSM 780 Confocal Microscope.

    Techniques: Immunolabeling, Staining, Microscopy

    Ataxin‐2 B accumulates in the nuclei and in the cortex of fat body cells. Immunolocalization using α‐dAtx2 B and anti‐rabbit IgG conjugated to Alexa 488 (green). Actin filaments were stained with phalloidin conjugated to Alexa 594 (red) and nuclei with 4′,6‐diamidino‐2‐phenylindole, fluorescent stain that binds strongly to A‐T rich regions in DNA (blue). The images were captured using the Zeiss LSM 780 confocal microscope.

    Journal: FEBS Open Bio

    Article Title: Drosophila ataxin‐2 gene encodes two differentially expressed isoforms and its function in larval fat body is crucial for development of peripheral tissues

    doi: 10.1002/2211-5463.12124

    Figure Lengend Snippet: Ataxin‐2 B accumulates in the nuclei and in the cortex of fat body cells. Immunolocalization using α‐dAtx2 B and anti‐rabbit IgG conjugated to Alexa 488 (green). Actin filaments were stained with phalloidin conjugated to Alexa 594 (red) and nuclei with 4′,6‐diamidino‐2‐phenylindole, fluorescent stain that binds strongly to A‐T rich regions in DNA (blue). The images were captured using the Zeiss LSM 780 confocal microscope.

    Article Snippet: The samples were examined using Leica TCS SP5 confocal microscope or Zeiss LSM 780 Confocal Microscope.

    Techniques: Staining, Microscopy

    Spheroid formation differs strongly between cell lines. (A) Relationship between the cell lines regarding origin of tissue, species and phenotype. (B) Spheroid formation with HC11 cells for 48 h. Images show transmission channel and fluorescence channel of cell nuclei tagged with H2B-eGFP. Wide-field fluorescence microscopy: Carl Zeiss Cell Observer Z.1, objective: 10×/NA 0.5, time-lapse: 48 h, interval: 30 min, Scale bar: 50 µm. (C) Dynamics of the projected area occupied by HC11, 4T1 and T47D cells in DMSO normalised to the area at time 0 h. Shaded regions represent the standard error of the mean (SEM). Number of independent experiments for HC11, 4T1 and T47D cells: 30, 40, 30. (D) The shrinkage rate was approximated by ΔNA/Δt, where ΔNA is the difference of the normalised area between two time points and Δt the time step. The transition between aggregation and compaction phase is reached when the shrinkage rate drops below 0.008. The growth phase is reached when the shrinkage rate is below zero. (E) Views along different directions show spheroid dimensions at 24 h, 48 h and 7 days of formation. The 4× magnified section shows a mitotic cell. Microscope: Zeiss LSM780, objective: 40×/NA 1.3 oil, Scale bar: 50 µm.

    Journal: Biology Open

    Article Title: E-cadherin, actin, microtubules and FAK dominate different spheroid formation phases and important elements of tissue integrity

    doi: 10.1242/bio.037051

    Figure Lengend Snippet: Spheroid formation differs strongly between cell lines. (A) Relationship between the cell lines regarding origin of tissue, species and phenotype. (B) Spheroid formation with HC11 cells for 48 h. Images show transmission channel and fluorescence channel of cell nuclei tagged with H2B-eGFP. Wide-field fluorescence microscopy: Carl Zeiss Cell Observer Z.1, objective: 10×/NA 0.5, time-lapse: 48 h, interval: 30 min, Scale bar: 50 µm. (C) Dynamics of the projected area occupied by HC11, 4T1 and T47D cells in DMSO normalised to the area at time 0 h. Shaded regions represent the standard error of the mean (SEM). Number of independent experiments for HC11, 4T1 and T47D cells: 30, 40, 30. (D) The shrinkage rate was approximated by ΔNA/Δt, where ΔNA is the difference of the normalised area between two time points and Δt the time step. The transition between aggregation and compaction phase is reached when the shrinkage rate drops below 0.008. The growth phase is reached when the shrinkage rate is below zero. (E) Views along different directions show spheroid dimensions at 24 h, 48 h and 7 days of formation. The 4× magnified section shows a mitotic cell. Microscope: Zeiss LSM780, objective: 40×/NA 1.3 oil, Scale bar: 50 µm.

    Article Snippet: Confocal laser scanning microscopy Immunostained spheroids were mounted in a drop of Mowiol on a cover glass and image stacks were acquired with a 2 µm spacing in a Zeiss LSM780 confocal microscope equipped with a 40×/NA 1.3 oil objective lens.

    Techniques: Transmission Assay, Fluorescence, Microscopy

    Extracellular matrix is expressed in cellular spheroids. (A,C) Amplification of human and murine Col1a1 and Fn1 mRNA. Amplicon length of human and mouse Col1a1 is 600 bp and 223 bp, respectively. Amplicon length of human and mouse Fn1 is 438 bp and 124 bp, respectively. BiP amplification was used as a control and had a fragment size of 560 bp. As positive control, RNA from murine mammary glands (MG), MCF10A epithelial breast cells and 22B endometriotic stromal cells was used. (A) RNA was extracted from cell lines in monolayer culture, or (C) from spheroids cultured for two or seven days. (B) Cell lines showed different behaviours in attaching to surfaces coated with ECM-components. (D) ECM components are expressed in spheroids within 24 h of formation. The different matrices show different patterns in the spheroids. Microscope: Zeiss LSM780, objective: 40×/1.3 oil, Scale bar: 50 µm. Col I, collagen I; Fn, fibronectin; B, BiP; C, collagen 1a1; F, fibronectin 1.

    Journal: Biology Open

    Article Title: E-cadherin, actin, microtubules and FAK dominate different spheroid formation phases and important elements of tissue integrity

    doi: 10.1242/bio.037051

    Figure Lengend Snippet: Extracellular matrix is expressed in cellular spheroids. (A,C) Amplification of human and murine Col1a1 and Fn1 mRNA. Amplicon length of human and mouse Col1a1 is 600 bp and 223 bp, respectively. Amplicon length of human and mouse Fn1 is 438 bp and 124 bp, respectively. BiP amplification was used as a control and had a fragment size of 560 bp. As positive control, RNA from murine mammary glands (MG), MCF10A epithelial breast cells and 22B endometriotic stromal cells was used. (A) RNA was extracted from cell lines in monolayer culture, or (C) from spheroids cultured for two or seven days. (B) Cell lines showed different behaviours in attaching to surfaces coated with ECM-components. (D) ECM components are expressed in spheroids within 24 h of formation. The different matrices show different patterns in the spheroids. Microscope: Zeiss LSM780, objective: 40×/1.3 oil, Scale bar: 50 µm. Col I, collagen I; Fn, fibronectin; B, BiP; C, collagen 1a1; F, fibronectin 1.

    Article Snippet: Confocal laser scanning microscopy Immunostained spheroids were mounted in a drop of Mowiol on a cover glass and image stacks were acquired with a 2 µm spacing in a Zeiss LSM780 confocal microscope equipped with a 40×/NA 1.3 oil objective lens.

    Techniques: Amplification, Positive Control, Cell Culture, Microscopy

    Anti-HIV-1 activity, multimerization, and subcellular localization of SWAP1 and SWAP2. ( a ) Effects of chimeric TRIMCyp proteins on HIV-1 infection. CRFK cells stably expressing pLPCX or indicated TRIMCyp proteins were either mock-infected or infected with serial dilutions of VSV-G pseudotyped HIV-1-GFP virus overnight. Forty-eight hours post-infection, infected cells were sorted by FACS. The results are representative of 4 independent experiments. ( b ) Multimerization of SWAP1 and SWAP2. Lysates from CRFK cells stably expressing TRIMCyp proteins were cross-linked using increasing concentrations of glutaraldehyde (0, 0.25, 0.5, 1, or 2 mM) for 3 minutes at 37°C. The reactions were terminated with the addition of 1 M Tris-HCl (pH 8.0), and cross-linked products were subjected to SDS-PAGE and visualized by Western blot using a mouse anti-HA antibody. Asterisk (*) indicates high-order multimers. Results are representative of 2 independent experiments. ( c ) Subcellular localization of SWAP1 and SWAP2. CRFK cells stably expressing TRIMCyp proteins were fixed and immunostained with a mouse anti-HA antibody (green). All images were obtained using an Olympus FluoView FV1000 confocal microscope. Nuclei were stained with DAPI (blue). The scale bar indicates 50 μm.

    Journal: PLoS ONE

    Article Title: The Effect of Exon 7 Deletion during the Evolution of TRIMCyp Fusion Proteins on Viral Restriction, Cytoplasmic Body Formation and Multimerization

    doi: 10.1371/journal.pone.0121666

    Figure Lengend Snippet: Anti-HIV-1 activity, multimerization, and subcellular localization of SWAP1 and SWAP2. ( a ) Effects of chimeric TRIMCyp proteins on HIV-1 infection. CRFK cells stably expressing pLPCX or indicated TRIMCyp proteins were either mock-infected or infected with serial dilutions of VSV-G pseudotyped HIV-1-GFP virus overnight. Forty-eight hours post-infection, infected cells were sorted by FACS. The results are representative of 4 independent experiments. ( b ) Multimerization of SWAP1 and SWAP2. Lysates from CRFK cells stably expressing TRIMCyp proteins were cross-linked using increasing concentrations of glutaraldehyde (0, 0.25, 0.5, 1, or 2 mM) for 3 minutes at 37°C. The reactions were terminated with the addition of 1 M Tris-HCl (pH 8.0), and cross-linked products were subjected to SDS-PAGE and visualized by Western blot using a mouse anti-HA antibody. Asterisk (*) indicates high-order multimers. Results are representative of 2 independent experiments. ( c ) Subcellular localization of SWAP1 and SWAP2. CRFK cells stably expressing TRIMCyp proteins were fixed and immunostained with a mouse anti-HA antibody (green). All images were obtained using an Olympus FluoView FV1000 confocal microscope. Nuclei were stained with DAPI (blue). The scale bar indicates 50 μm.

    Article Snippet: Images were obtained using an Olympus FluoView FV1000 confocal microscope.

    Techniques: Activity Assay, Infection, Stable Transfection, Expressing, FACS, SDS Page, Western Blot, Microscopy, Staining

    Anti-HIV-1 activity, multimerization, and subcellular localization of SWAP3, SWAP4, and SWAP5. ( a ) Effects of chimeric TRIMCyp proteins on HIV-1 infection. CRFK cells stably expressing pLPCX or TRIMCyp proteins were mock-infected or infected with serial dilutions of VSV-G pseudotyped HIV-1-GFP virus overnight. Forty-eight hours post-infection, infected cells were counted by FACS. The results are representative of 3 independent experiments. ( b ) Subcellular localization of SWAP3, SWAP4, and SWAP5. CRFK cells stably expressing TRIMCyp proteins were fixed and immunostained using a mouse anti-HA antibody (green). All images were obtained using an Olympus FluoView FV1000 confocal microscope. Nuclei were stained with DAPI (blue). The scale bar indicates 50 μm. ( c ) Multimerization of SWAP3, SWAP4, and SWAP5. Lysates from CRFK cells stably expressing the indicated TRIMCyp proteins were cross-linked with increasing concentrations of glutaraldehyde (0, 0.25, 0.5, 1, or 2 mM) for 3 minutes at 37°C. The reaction was terminated with the addition of 1 M Tris-HCl (pH 8.0), and cross-linked products were subjected to SDS-PAGE and visualized by Western blot using a mouse anti-HA antibody. Asterisk (*) indicates high-order multimers. The results are representative of 2 independent experiments.

    Journal: PLoS ONE

    Article Title: The Effect of Exon 7 Deletion during the Evolution of TRIMCyp Fusion Proteins on Viral Restriction, Cytoplasmic Body Formation and Multimerization

    doi: 10.1371/journal.pone.0121666

    Figure Lengend Snippet: Anti-HIV-1 activity, multimerization, and subcellular localization of SWAP3, SWAP4, and SWAP5. ( a ) Effects of chimeric TRIMCyp proteins on HIV-1 infection. CRFK cells stably expressing pLPCX or TRIMCyp proteins were mock-infected or infected with serial dilutions of VSV-G pseudotyped HIV-1-GFP virus overnight. Forty-eight hours post-infection, infected cells were counted by FACS. The results are representative of 3 independent experiments. ( b ) Subcellular localization of SWAP3, SWAP4, and SWAP5. CRFK cells stably expressing TRIMCyp proteins were fixed and immunostained using a mouse anti-HA antibody (green). All images were obtained using an Olympus FluoView FV1000 confocal microscope. Nuclei were stained with DAPI (blue). The scale bar indicates 50 μm. ( c ) Multimerization of SWAP3, SWAP4, and SWAP5. Lysates from CRFK cells stably expressing the indicated TRIMCyp proteins were cross-linked with increasing concentrations of glutaraldehyde (0, 0.25, 0.5, 1, or 2 mM) for 3 minutes at 37°C. The reaction was terminated with the addition of 1 M Tris-HCl (pH 8.0), and cross-linked products were subjected to SDS-PAGE and visualized by Western blot using a mouse anti-HA antibody. Asterisk (*) indicates high-order multimers. The results are representative of 2 independent experiments.

    Article Snippet: Images were obtained using an Olympus FluoView FV1000 confocal microscope.

    Techniques: Activity Assay, Infection, Stable Transfection, Expressing, FACS, Microscopy, Staining, SDS Page, Western Blot

    omTRIMCyp and npmTRIMCyp present different status of cytoplasmic bodies and multimerization. ( a ) The subcellular localization of omTRIMCyp and npmTRIMCyp was examined. CRFK cells stably expressing pLPCX or indicated TRIMCyp proteins were fixed and immunostained with a mouse anti-HA antibody (green). All images were obtained using an Olympus FluoView FV1000 confocal microscope. Nuclei were stained with DAPI (blue). The scale bar indicates 50 μm. ( b ) Multimerization of omTRIMCyp and npmTRIMCyp proteins was examined. Lysates from CRFK cells stably expressing TRIMCyp proteins were cross-linked with increasing concentrations of glutaraldehyde (0, 0.25, 0.5, 1, or 2 mM) for 3 minutes at 37°C. The reactions were terminated with the addition of 1 M Tris-HCl (pH 8.0), and cross-linked products were subjected to SDS-PAGE and visualized by Western blot using a mouse anti-HA antibody. Asterisk (*) indicates high-order multimers.

    Journal: PLoS ONE

    Article Title: The Effect of Exon 7 Deletion during the Evolution of TRIMCyp Fusion Proteins on Viral Restriction, Cytoplasmic Body Formation and Multimerization

    doi: 10.1371/journal.pone.0121666

    Figure Lengend Snippet: omTRIMCyp and npmTRIMCyp present different status of cytoplasmic bodies and multimerization. ( a ) The subcellular localization of omTRIMCyp and npmTRIMCyp was examined. CRFK cells stably expressing pLPCX or indicated TRIMCyp proteins were fixed and immunostained with a mouse anti-HA antibody (green). All images were obtained using an Olympus FluoView FV1000 confocal microscope. Nuclei were stained with DAPI (blue). The scale bar indicates 50 μm. ( b ) Multimerization of omTRIMCyp and npmTRIMCyp proteins was examined. Lysates from CRFK cells stably expressing TRIMCyp proteins were cross-linked with increasing concentrations of glutaraldehyde (0, 0.25, 0.5, 1, or 2 mM) for 3 minutes at 37°C. The reactions were terminated with the addition of 1 M Tris-HCl (pH 8.0), and cross-linked products were subjected to SDS-PAGE and visualized by Western blot using a mouse anti-HA antibody. Asterisk (*) indicates high-order multimers.

    Article Snippet: Images were obtained using an Olympus FluoView FV1000 confocal microscope.

    Techniques: Stable Transfection, Expressing, Microscopy, Staining, SDS Page, Western Blot

    Optical sections of an isolated salt gland of  A .  officinalis . Sections (A-E) were taken in the fluorescent mode to show the side views of a salt gland. Details within cells could be observed due to the green autofluorescent nature of the salt gland. Note the presence of strongly autofluorescent structures (arrows) in the middle or periphery of some secretory cells. (F) Corresponding bright-field image of (C) taken together with overlapping image recorded simultaneously in the transmission and fluorescent modes. Optical sections were taken with Zeiss LSM 510. Excitation and emission wavelengths were 488 nm (10%) and  > 530 nm, respectively. Bars = 5 μm.

    Journal: Plant Methods

    Article Title: A simple, rapid method to isolate salt glands for three-dimensional visualization, fluorescence imaging and cytological studies

    doi: 10.1186/1746-4811-6-24

    Figure Lengend Snippet: Optical sections of an isolated salt gland of A . officinalis . Sections (A-E) were taken in the fluorescent mode to show the side views of a salt gland. Details within cells could be observed due to the green autofluorescent nature of the salt gland. Note the presence of strongly autofluorescent structures (arrows) in the middle or periphery of some secretory cells. (F) Corresponding bright-field image of (C) taken together with overlapping image recorded simultaneously in the transmission and fluorescent modes. Optical sections were taken with Zeiss LSM 510. Excitation and emission wavelengths were 488 nm (10%) and > 530 nm, respectively. Bars = 5 μm.

    Article Snippet: Confocal Laser Scanning Microscopy Observations of isolated salt glands were performed with a Zeiss LSM 510 laser scanning confocal microscope.

    Techniques: Isolation, Transmission Assay

    Confocal microscopic images of isolated A . officinalis salt glands showing the degree of red autofluorescence . (A) Image of isolated salt glands showing red autofluorescence (faintly visible). (B) Image of isolated salt glands captured simultaneously in the transmission and fluorescent modes. Excitation wavelength was 543 nm (100%) and signals in the red wavelength range were captured with Zeiss LSM 510 using high pass filter 560 nm. Bars = 20 μm.

    Journal: Plant Methods

    Article Title: A simple, rapid method to isolate salt glands for three-dimensional visualization, fluorescence imaging and cytological studies

    doi: 10.1186/1746-4811-6-24

    Figure Lengend Snippet: Confocal microscopic images of isolated A . officinalis salt glands showing the degree of red autofluorescence . (A) Image of isolated salt glands showing red autofluorescence (faintly visible). (B) Image of isolated salt glands captured simultaneously in the transmission and fluorescent modes. Excitation wavelength was 543 nm (100%) and signals in the red wavelength range were captured with Zeiss LSM 510 using high pass filter 560 nm. Bars = 20 μm.

    Article Snippet: Confocal Laser Scanning Microscopy Observations of isolated salt glands were performed with a Zeiss LSM 510 laser scanning confocal microscope.

    Techniques: Isolation, Transmission Assay

    Comparison of microtome salt gland section with isolated salt gland of  A. officinalis . (A) Microtome section of pre-fixed paraffin-embedded leaf specimen showing cuticle envelope (cu) surrounding the salt gland and above the epidermal cells (epc). Bar = 20 μm. (B) Three-dimensional image of isolated salt gland (side view) showing 4 secretory cells (sc) above the stalk cell (st). The three-dimensional image was constructed using Imaris™ (beta) 3.0, based on stacked images captured with Zeiss LSM 510. Excitation and emission wavelengths were 488 nm (43%) and  > 505 nm, respectively. Bar = 10 μm. Note the presence of two collecting cells (cc) in (A) which are lacking in the isolated salt gland (B).

    Journal: Plant Methods

    Article Title: A simple, rapid method to isolate salt glands for three-dimensional visualization, fluorescence imaging and cytological studies

    doi: 10.1186/1746-4811-6-24

    Figure Lengend Snippet: Comparison of microtome salt gland section with isolated salt gland of A. officinalis . (A) Microtome section of pre-fixed paraffin-embedded leaf specimen showing cuticle envelope (cu) surrounding the salt gland and above the epidermal cells (epc). Bar = 20 μm. (B) Three-dimensional image of isolated salt gland (side view) showing 4 secretory cells (sc) above the stalk cell (st). The three-dimensional image was constructed using Imaris™ (beta) 3.0, based on stacked images captured with Zeiss LSM 510. Excitation and emission wavelengths were 488 nm (43%) and > 505 nm, respectively. Bar = 10 μm. Note the presence of two collecting cells (cc) in (A) which are lacking in the isolated salt gland (B).

    Article Snippet: Confocal Laser Scanning Microscopy Observations of isolated salt glands were performed with a Zeiss LSM 510 laser scanning confocal microscope.

    Techniques: Isolation, Construct

    Optical sections of a side view salt gland showing the distribution of nuclei . (A-H) Positions of nuclei (purple pseudo-colour; 1-6) at different focal planes. (I) Corresponding bright-field image of the same salt gland under study. The salt gland was stained with Coulter ®  DNA-Prep Stain containing 8 μg ml -1  PI and images were captured using Zeiss LSM 510. Excitation and emission wavelengths were 543 nm and  > 560 nm, respectively. Bars = 5 μm.

    Journal: Plant Methods

    Article Title: A simple, rapid method to isolate salt glands for three-dimensional visualization, fluorescence imaging and cytological studies

    doi: 10.1186/1746-4811-6-24

    Figure Lengend Snippet: Optical sections of a side view salt gland showing the distribution of nuclei . (A-H) Positions of nuclei (purple pseudo-colour; 1-6) at different focal planes. (I) Corresponding bright-field image of the same salt gland under study. The salt gland was stained with Coulter ® DNA-Prep Stain containing 8 μg ml -1 PI and images were captured using Zeiss LSM 510. Excitation and emission wavelengths were 543 nm and > 560 nm, respectively. Bars = 5 μm.

    Article Snippet: Confocal Laser Scanning Microscopy Observations of isolated salt glands were performed with a Zeiss LSM 510 laser scanning confocal microscope.

    Techniques: Staining

    Isolated salt glands of  A .  officinalis  showing their intrinsic autofluorescent nature . (A-C) Side view of salt gland. Note the presence of 2 secretory cells (sc) above the stalk cell (st). (D-F) Top view of salt gland with a total of 7 secretory cells (green patches) observed. The outlines of these cells were emphasized by their autofluorescent nature. Excitation wavelength was 488 nm (8-10%) and optical sections were taken using Zeiss LSM 510. Images from optical sectioning were stacked to give a clearer picture of the salt glands. Bars = 5 μm.

    Journal: Plant Methods

    Article Title: A simple, rapid method to isolate salt glands for three-dimensional visualization, fluorescence imaging and cytological studies

    doi: 10.1186/1746-4811-6-24

    Figure Lengend Snippet: Isolated salt glands of A . officinalis showing their intrinsic autofluorescent nature . (A-C) Side view of salt gland. Note the presence of 2 secretory cells (sc) above the stalk cell (st). (D-F) Top view of salt gland with a total of 7 secretory cells (green patches) observed. The outlines of these cells were emphasized by their autofluorescent nature. Excitation wavelength was 488 nm (8-10%) and optical sections were taken using Zeiss LSM 510. Images from optical sectioning were stacked to give a clearer picture of the salt glands. Bars = 5 μm.

    Article Snippet: Confocal Laser Scanning Microscopy Observations of isolated salt glands were performed with a Zeiss LSM 510 laser scanning confocal microscope.

    Techniques: Isolation

    Three-dimensional images of isolated  A .  officinalis  salt gland . Top view (A) and side view (B) of the same salt gland showing 8 secretory cells (arrows). Optical sections were first taken using Zeiss LSM 510. Excitation and emission wavelengths were 488 nm (12%) and  > 505 nm, respectively. Each three-dimensional image was constructed from the stacked images using Imaris™ (beta) 3.0. Bars = 10 μm.

    Journal: Plant Methods

    Article Title: A simple, rapid method to isolate salt glands for three-dimensional visualization, fluorescence imaging and cytological studies

    doi: 10.1186/1746-4811-6-24

    Figure Lengend Snippet: Three-dimensional images of isolated A . officinalis salt gland . Top view (A) and side view (B) of the same salt gland showing 8 secretory cells (arrows). Optical sections were first taken using Zeiss LSM 510. Excitation and emission wavelengths were 488 nm (12%) and > 505 nm, respectively. Each three-dimensional image was constructed from the stacked images using Imaris™ (beta) 3.0. Bars = 10 μm.

    Article Snippet: Confocal Laser Scanning Microscopy Observations of isolated salt glands were performed with a Zeiss LSM 510 laser scanning confocal microscope.

    Techniques: Isolation, Construct

    Double staining for FASN and NS3. HEK293T/17 cells were pre-treated with 10 μM orlistat or not treated before mock infection or infection with DENV 2. At ( a ) 24 or ( b ) 36 h.p.i cells were fixed and stained to show the FASN ( green ), DENV 2 NS3 protein ( red ) and nuclei ( blue ). Cells were examined under an Olympus FluoView 1000 confocal microscope with 60× magnification. Representative, non-contrast adjusted merged images are shown

    Journal: Virology Journal

    Article Title: Involvement of fatty acid synthase in dengue virus infection

    doi: 10.1186/s12985-017-0685-9

    Figure Lengend Snippet: Double staining for FASN and NS3. HEK293T/17 cells were pre-treated with 10 μM orlistat or not treated before mock infection or infection with DENV 2. At ( a ) 24 or ( b ) 36 h.p.i cells were fixed and stained to show the FASN ( green ), DENV 2 NS3 protein ( red ) and nuclei ( blue ). Cells were examined under an Olympus FluoView 1000 confocal microscope with 60× magnification. Representative, non-contrast adjusted merged images are shown

    Article Snippet: Signals were observed under an Olympus FluoView 1000 confocal microscope (Olympus Corporation, Shinjuku-ku, Tokyo, Japan) equipped with Olympus FluoView Software v. 1.6.

    Techniques: Double Staining, Infection, Staining, Microscopy

    Effect of orlistat on DENV 2 NS1 expression. HEK293T/17 cells were pre-treated with 10 μM orlistat or not treated before mock infection or infection with DENV 2. At ( a ) 24 or ( b ) 36 h.p.i cells were fixed and stained to show the nucleus ( blue ) and DENV 2 NS1 protein ( red ). Cells were examined under an Olympus FluoView 1000 confocal microscope with 60× magnification. Representative, non-contrast adjusted merged images are shown

    Journal: Virology Journal

    Article Title: Involvement of fatty acid synthase in dengue virus infection

    doi: 10.1186/s12985-017-0685-9

    Figure Lengend Snippet: Effect of orlistat on DENV 2 NS1 expression. HEK293T/17 cells were pre-treated with 10 μM orlistat or not treated before mock infection or infection with DENV 2. At ( a ) 24 or ( b ) 36 h.p.i cells were fixed and stained to show the nucleus ( blue ) and DENV 2 NS1 protein ( red ). Cells were examined under an Olympus FluoView 1000 confocal microscope with 60× magnification. Representative, non-contrast adjusted merged images are shown

    Article Snippet: Signals were observed under an Olympus FluoView 1000 confocal microscope (Olympus Corporation, Shinjuku-ku, Tokyo, Japan) equipped with Olympus FluoView Software v. 1.6.

    Techniques: Expressing, Infection, Staining, Microscopy