african green monkey kidney ma104 cells  (ATCC)


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    Structured Review

    ATCC african green monkey kidney ma104 cells
    RIG-I is down-regulated by NSP1 at the protein level but is proteasome-independent . (A, B) Western blot analysis of RIG-I down-regulation by NSP1. 293FT cells were transfected with increased amount of pEGFP-OSU NSP1 (A) or pEGFP-SA11 NSP1 (B) and plasmid encoding Myc-RIG-I. Cell extracts were prepared 48 h post-transfection. Immunoblots were probed with anti-Myc monoclonal antibody to detect RIG-I (top panel). β-actin was used as a loading control (bottom panel). (C) Transcription level of RIG-I at different time points after transfection. 293FT cells were co-transfected with SA11-NSP1 and RIG-I plasmids. At different time points after transfection, total RNA extracted from cells was subjected to RT-PCR amplification and electrophoresis for RIG-I, NSP1 and GAPDH (inner control) mRNAs. (D, E) RIG-I is degraded in rotavirus infected cells. <t>MA104</t> cells were infected with rotavirus SA11 at a m.o.i. of 0.1. Cell extracts were prepared at 0, 4, 8, 12, 24 and 36 h post-infection (p.i). RIG-I protein levels at each time point p.i. were determined by Western blot analyses using an anti-RIG-I antibody. The viral protein VP6 was used as an indicator for rotavirus infection. β-actin was used as a loading control. RIG-I, NSP1, VP6 and GAPDH mRNAs were also checked in parallel for evaluating the transcription level (E). (F, G) Effects of a proteasome inhibitor on NSP1 mediated RIG-I down-regulation. 293FT cells were transfected with Myc-RIG-I, pEGFP-OSU NSP1 (F) or pEGFP-SA11 NSP1 (G). The cells were treated with the proteasome inhibitor MG132 or an equivalent volume of DMSO as described in Methods. Lysates were prepared 36-48 h post-transfection. Immunoblots were probed with anti-Myc to detect myc-tagged RIG-I.
    African Green Monkey Kidney Ma104 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 97/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Rotavirus nonstructural protein 1 antagonizes innate immune response by interacting with retinoic acid inducible gene I"

    Article Title: Rotavirus nonstructural protein 1 antagonizes innate immune response by interacting with retinoic acid inducible gene I

    Journal: Virology Journal

    doi: 10.1186/1743-422X-8-526

    RIG-I is down-regulated by NSP1 at the protein level but is proteasome-independent . (A, B) Western blot analysis of RIG-I down-regulation by NSP1. 293FT cells were transfected with increased amount of pEGFP-OSU NSP1 (A) or pEGFP-SA11 NSP1 (B) and plasmid encoding Myc-RIG-I. Cell extracts were prepared 48 h post-transfection. Immunoblots were probed with anti-Myc monoclonal antibody to detect RIG-I (top panel). β-actin was used as a loading control (bottom panel). (C) Transcription level of RIG-I at different time points after transfection. 293FT cells were co-transfected with SA11-NSP1 and RIG-I plasmids. At different time points after transfection, total RNA extracted from cells was subjected to RT-PCR amplification and electrophoresis for RIG-I, NSP1 and GAPDH (inner control) mRNAs. (D, E) RIG-I is degraded in rotavirus infected cells. MA104 cells were infected with rotavirus SA11 at a m.o.i. of 0.1. Cell extracts were prepared at 0, 4, 8, 12, 24 and 36 h post-infection (p.i). RIG-I protein levels at each time point p.i. were determined by Western blot analyses using an anti-RIG-I antibody. The viral protein VP6 was used as an indicator for rotavirus infection. β-actin was used as a loading control. RIG-I, NSP1, VP6 and GAPDH mRNAs were also checked in parallel for evaluating the transcription level (E). (F, G) Effects of a proteasome inhibitor on NSP1 mediated RIG-I down-regulation. 293FT cells were transfected with Myc-RIG-I, pEGFP-OSU NSP1 (F) or pEGFP-SA11 NSP1 (G). The cells were treated with the proteasome inhibitor MG132 or an equivalent volume of DMSO as described in Methods. Lysates were prepared 36-48 h post-transfection. Immunoblots were probed with anti-Myc to detect myc-tagged RIG-I.
    Figure Legend Snippet: RIG-I is down-regulated by NSP1 at the protein level but is proteasome-independent . (A, B) Western blot analysis of RIG-I down-regulation by NSP1. 293FT cells were transfected with increased amount of pEGFP-OSU NSP1 (A) or pEGFP-SA11 NSP1 (B) and plasmid encoding Myc-RIG-I. Cell extracts were prepared 48 h post-transfection. Immunoblots were probed with anti-Myc monoclonal antibody to detect RIG-I (top panel). β-actin was used as a loading control (bottom panel). (C) Transcription level of RIG-I at different time points after transfection. 293FT cells were co-transfected with SA11-NSP1 and RIG-I plasmids. At different time points after transfection, total RNA extracted from cells was subjected to RT-PCR amplification and electrophoresis for RIG-I, NSP1 and GAPDH (inner control) mRNAs. (D, E) RIG-I is degraded in rotavirus infected cells. MA104 cells were infected with rotavirus SA11 at a m.o.i. of 0.1. Cell extracts were prepared at 0, 4, 8, 12, 24 and 36 h post-infection (p.i). RIG-I protein levels at each time point p.i. were determined by Western blot analyses using an anti-RIG-I antibody. The viral protein VP6 was used as an indicator for rotavirus infection. β-actin was used as a loading control. RIG-I, NSP1, VP6 and GAPDH mRNAs were also checked in parallel for evaluating the transcription level (E). (F, G) Effects of a proteasome inhibitor on NSP1 mediated RIG-I down-regulation. 293FT cells were transfected with Myc-RIG-I, pEGFP-OSU NSP1 (F) or pEGFP-SA11 NSP1 (G). The cells were treated with the proteasome inhibitor MG132 or an equivalent volume of DMSO as described in Methods. Lysates were prepared 36-48 h post-transfection. Immunoblots were probed with anti-Myc to detect myc-tagged RIG-I.

    Techniques Used: Western Blot, Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Amplification, Electrophoresis, Infection

    2) Product Images from "Cell-Cell Fusion Induced by Measles Virus Amplifies the Type I Interferon Response ▿Cell-Cell Fusion Induced by Measles Virus Amplifies the Type I Interferon Response ▿ †"

    Article Title: Cell-Cell Fusion Induced by Measles Virus Amplifies the Type I Interferon Response ▿Cell-Cell Fusion Induced by Measles Virus Amplifies the Type I Interferon Response ▿ †

    Journal:

    doi: 10.1128/JVI.00078-07

    Reciprocal trans -complementation of RIG-I- and IFN-β-deficient cells by MeV-induced fusion. RIG-I-deficient Huh7.5 or IFN-β-deficient Vero cells were infected with MeV at an MOI of 1 and cocultured 8 h later with uninfected Vero and Huh7.5
    Figure Legend Snippet: Reciprocal trans -complementation of RIG-I- and IFN-β-deficient cells by MeV-induced fusion. RIG-I-deficient Huh7.5 or IFN-β-deficient Vero cells were infected with MeV at an MOI of 1 and cocultured 8 h later with uninfected Vero and Huh7.5

    Techniques Used: Infection

    3) Product Images from "VP1, the Putative RNA-Dependent RNA Polymerase of Infectious Bursal Disease Virus, Forms Complexes with the Capsid Protein VP3, Leading to Efficient Encapsidation into Virus-Like Particles"

    Article Title: VP1, the Putative RNA-Dependent RNA Polymerase of Infectious Bursal Disease Virus, Forms Complexes with the Capsid Protein VP3, Leading to Efficient Encapsidation into Virus-Like Particles

    Journal: Journal of Virology

    doi:

    Characterization of the expression of IBDV VP1 in cells infected with the rVV VT7/VP1. (A) BSC-1 cells infected with VT7/VP1 or the parental virus VT7LacOI, either treated (+) or untreated (−) with the inducer IPTG, were metabolically labelled with [ 35 S]methionine. CEF mock-infected (Mock) or infected with IBDV (IBDV) were metabolically labelled with [ 35 S]methionine at 48 h p.i. Protein samples from the different cultures were analyzed by SDS-PAGE. After electrophoresis, the gel was electroblotted onto nitrocellulose. The filter was air-dried and subjected to autoradiography. The positions of molecular weight markers are indicated (Mw). The positions of bands corresponding to the VP1 polypeptide are indicated by open arrowheads. (B) Western blot analysis of proteins expressed in cells infected with the rVV VT7/VP1 and CEF infected with IBDV. To determine the position of the VP1 polypeptide, after autoradiography, the nitrocellulose filter was rehydrated and incubated with a rabbit anti-VP1 antisera followed by the addition of horseradish peroxidase-conjugated goat anti-rabbit Ig. The signal was detected by ECL.
    Figure Legend Snippet: Characterization of the expression of IBDV VP1 in cells infected with the rVV VT7/VP1. (A) BSC-1 cells infected with VT7/VP1 or the parental virus VT7LacOI, either treated (+) or untreated (−) with the inducer IPTG, were metabolically labelled with [ 35 S]methionine. CEF mock-infected (Mock) or infected with IBDV (IBDV) were metabolically labelled with [ 35 S]methionine at 48 h p.i. Protein samples from the different cultures were analyzed by SDS-PAGE. After electrophoresis, the gel was electroblotted onto nitrocellulose. The filter was air-dried and subjected to autoradiography. The positions of molecular weight markers are indicated (Mw). The positions of bands corresponding to the VP1 polypeptide are indicated by open arrowheads. (B) Western blot analysis of proteins expressed in cells infected with the rVV VT7/VP1 and CEF infected with IBDV. To determine the position of the VP1 polypeptide, after autoradiography, the nitrocellulose filter was rehydrated and incubated with a rabbit anti-VP1 antisera followed by the addition of horseradish peroxidase-conjugated goat anti-rabbit Ig. The signal was detected by ECL.

    Techniques Used: Expressing, Infection, Metabolic Labelling, SDS Page, Electrophoresis, Autoradiography, Molecular Weight, Western Blot, Incubation

    4) Product Images from "Absence of an N-Linked Glycosylation Motif in the Glycoprotein of the Live-Attenuated Argentine Hemorrhagic Fever Vaccine, Candid #1, Results in Its Improper Processing, and Reduced Surface Expression"

    Article Title: Absence of an N-Linked Glycosylation Motif in the Glycoprotein of the Live-Attenuated Argentine Hemorrhagic Fever Vaccine, Candid #1, Results in Its Improper Processing, and Reduced Surface Expression

    Journal: Frontiers in Cellular and Infection Microbiology

    doi: 10.3389/fcimb.2017.00020

    N-linked glycosylation status of JUNV GPC. (A) Lysates of Vero cells infected at an MOI of 5 with either rRom (Romero) or rCan (Candid) were treated with PNGase F to remove N-linked oligosaccharides from protein backbone. The GPC expression profiles were analyzed by Western blotting using an anti-G2 antibody. (B) Activation of the UPR in HEK 293 cells expressing GPC of JUNV strains differing in in vivo attenuation status. Cells were transfected with equal amounts of expression plasmids for the GPC of Rom (Romero), Can (Candid), Can predecessor strains XJ13 and XJ44, and the GPC of Can and Rom bearing I427F (CanI427F) and F427I (RomF427I) amino acid changes in the G2 subunit, and A168T and T168A amino acid changes in the G1 subunit (C) , respectively. Cell lysates were harvested at 24 h post-transfection and divided into two equal aliquots that were either mock-treated (top panels) or treated with PNGase F (PNGase F treated). Expression of viral GPC and the UPR marker BiP was analyzed by Western blotting. 293, HEK 293 cells transfected with a GFP expressing construct that shares the same plasmid backbone with the expression constructs for JUNV GPC. Densitometry was calculated using AlphaEase TM in order to determine saturation of each band. Band saturation of the selected area is represented on a scale from 0 to 255. (C) GPC expression profiles in cells with or without the T168A substitution. Cells were transfected with equal amounts of each plasmid and allowed to incubate for 36 h. HEK 293 cell were transfected with a GFP plasmid containing the same backbone as the GPC expression plasmids. The cells were lysed and analyzed by Western blotting.
    Figure Legend Snippet: N-linked glycosylation status of JUNV GPC. (A) Lysates of Vero cells infected at an MOI of 5 with either rRom (Romero) or rCan (Candid) were treated with PNGase F to remove N-linked oligosaccharides from protein backbone. The GPC expression profiles were analyzed by Western blotting using an anti-G2 antibody. (B) Activation of the UPR in HEK 293 cells expressing GPC of JUNV strains differing in in vivo attenuation status. Cells were transfected with equal amounts of expression plasmids for the GPC of Rom (Romero), Can (Candid), Can predecessor strains XJ13 and XJ44, and the GPC of Can and Rom bearing I427F (CanI427F) and F427I (RomF427I) amino acid changes in the G2 subunit, and A168T and T168A amino acid changes in the G1 subunit (C) , respectively. Cell lysates were harvested at 24 h post-transfection and divided into two equal aliquots that were either mock-treated (top panels) or treated with PNGase F (PNGase F treated). Expression of viral GPC and the UPR marker BiP was analyzed by Western blotting. 293, HEK 293 cells transfected with a GFP expressing construct that shares the same plasmid backbone with the expression constructs for JUNV GPC. Densitometry was calculated using AlphaEase TM in order to determine saturation of each band. Band saturation of the selected area is represented on a scale from 0 to 255. (C) GPC expression profiles in cells with or without the T168A substitution. Cells were transfected with equal amounts of each plasmid and allowed to incubate for 36 h. HEK 293 cell were transfected with a GFP plasmid containing the same backbone as the GPC expression plasmids. The cells were lysed and analyzed by Western blotting.

    Techniques Used: Gel Permeation Chromatography, Infection, Expressing, Western Blot, Activation Assay, In Vivo, Transfection, Marker, Construct, Plasmid Preparation

    5) Product Images from "Sequestering of Rac by the Yersinia Effector YopO Blocks Fc? Receptor-mediated Phagocytosis *"

    Article Title: Sequestering of Rac by the Yersinia Effector YopO Blocks Fc? Receptor-mediated Phagocytosis *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.071035

    Domain analysis of YopO. The GDI-like domain (RBD) is the main contributor to anti-phagocytosis. A , COS-7 cells were co-transfected with FcγRIIA and GFP-tagged constructs as indicated. After 24 h, cells were challenged with IgG-opsonized sRBC
    Figure Legend Snippet: Domain analysis of YopO. The GDI-like domain (RBD) is the main contributor to anti-phagocytosis. A , COS-7 cells were co-transfected with FcγRIIA and GFP-tagged constructs as indicated. After 24 h, cells were challenged with IgG-opsonized sRBC

    Techniques Used: Transfection, Construct

    YopO does not affect Rac recruitment to FcγR-phagocytic cups. A , COS-7 cells were transiently transfected with FcγRIIA wild-type ( wt ) or mutant FcγRIIA ( Y2F ) and either GFP or GFP-YopOwt and then challenged with IgG-opsonized sRBC
    Figure Legend Snippet: YopO does not affect Rac recruitment to FcγR-phagocytic cups. A , COS-7 cells were transiently transfected with FcγRIIA wild-type ( wt ) or mutant FcγRIIA ( Y2F ) and either GFP or GFP-YopOwt and then challenged with IgG-opsonized sRBC

    Techniques Used: Transfection, Mutagenesis

    Unlike RhoGDI, YopO RBD preferentially binds Rac from cell lysates. J774A.1 ( A ) or COS-7 ( B ) lysates were incubated with GST-YopO RBD immobilized on glutathione-Sepharose beads, and bead eluates were resolved by SDS-PAGE and Western blotting, probing
    Figure Legend Snippet: Unlike RhoGDI, YopO RBD preferentially binds Rac from cell lysates. J774A.1 ( A ) or COS-7 ( B ) lysates were incubated with GST-YopO RBD immobilized on glutathione-Sepharose beads, and bead eluates were resolved by SDS-PAGE and Western blotting, probing

    Techniques Used: Incubation, SDS Page, Western Blot

    GDI-free pool of Rac molecules exists at the plasma membrane of resting cells. J774A.1 ( A ) and COS-7 ( B ) lysates were separated into membrane ( M ) and cytosol ( C ) fractions as described under “Experimental Procedures.” After SDS-PAGE, blots
    Figure Legend Snippet: GDI-free pool of Rac molecules exists at the plasma membrane of resting cells. J774A.1 ( A ) and COS-7 ( B ) lysates were separated into membrane ( M ) and cytosol ( C ) fractions as described under “Experimental Procedures.” After SDS-PAGE, blots

    Techniques Used: SDS Page

    YopO blocks Rac activation during FcγR phagocytosis. A , COS-7 cells were transiently transfected with FcγRIIA and either GFP or GFP-YopOwt and then challenged with IgG-opsonized sRBC for 10 min at 37 °C after synchronization at
    Figure Legend Snippet: YopO blocks Rac activation during FcγR phagocytosis. A , COS-7 cells were transiently transfected with FcγRIIA and either GFP or GFP-YopOwt and then challenged with IgG-opsonized sRBC for 10 min at 37 °C after synchronization at

    Techniques Used: Activation Assay, Transfection

    6) Product Images from "c-Myb Binding Sites in Haematopoietic Chromatin Landscapes"

    Article Title: c-Myb Binding Sites in Haematopoietic Chromatin Landscapes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0133280

    c-Myb enhances transcription from genomic elements with c-Myb footprints. Luciferase-based reporter assay to study the responsiveness of genomic regions, with one or more c-MYB footprints mapped. (A) Representative Immunoblot of CV1 cells transfected with reporter plasmid and-/+ c-Myb. (B) Map of the pGL4.26 vector used for the luciferase assays. The grey box illustrates the genomic fragment containing a c-Myb footprint or control region. The black box illustrates the minimal promoter. (C) Positive control with three MREs. (D-E) Negative controls, (F-K) genomic loci identified to contain c-Myb footprints. The upper panels show the genomic region in the UCSC browser (hg19) presenting DNase I signals, c-Myb footprints and oligos for selected region. The coordinates for the c-Myb footprint are shown above the illustration. The values are the average from three independent experiments-/+ SEM.
    Figure Legend Snippet: c-Myb enhances transcription from genomic elements with c-Myb footprints. Luciferase-based reporter assay to study the responsiveness of genomic regions, with one or more c-MYB footprints mapped. (A) Representative Immunoblot of CV1 cells transfected with reporter plasmid and-/+ c-Myb. (B) Map of the pGL4.26 vector used for the luciferase assays. The grey box illustrates the genomic fragment containing a c-Myb footprint or control region. The black box illustrates the minimal promoter. (C) Positive control with three MREs. (D-E) Negative controls, (F-K) genomic loci identified to contain c-Myb footprints. The upper panels show the genomic region in the UCSC browser (hg19) presenting DNase I signals, c-Myb footprints and oligos for selected region. The coordinates for the c-Myb footprint are shown above the illustration. The values are the average from three independent experiments-/+ SEM.

    Techniques Used: Luciferase, Reporter Assay, Transfection, Plasmid Preparation, Positive Control

    7) Product Images from "LncRNA KCNA2-AS regulates spinal astrocyte activation through STAT3 to affect postherpetic neuralgia"

    Article Title: LncRNA KCNA2-AS regulates spinal astrocyte activation through STAT3 to affect postherpetic neuralgia

    Journal: Molecular Medicine

    doi: 10.1186/s10020-020-00232-9

    NO expression was increased in the spinal cord of PHN model rats. The PHN rat model was constructed with a consistent approach to the above description. The sham group rats were injected with uninfected CV-1 cells as control. The experiment lasted 4 weeks, and 5 rats were sacrificed in each group every other week. a The level of nitric oxide (NO) was detected. b Spinal cord tissues were collected from the PHN group and sham group at 1 w, 2 w and 4 w, immunohistochemical staining was used to detect the level of NO. c Spinal cord tissues were collected from the PHN group and sham group at 1 w, 2 w, and 4 w, respectively, and immunofluorescence staining was performed by using NOS/GFAP. ** P
    Figure Legend Snippet: NO expression was increased in the spinal cord of PHN model rats. The PHN rat model was constructed with a consistent approach to the above description. The sham group rats were injected with uninfected CV-1 cells as control. The experiment lasted 4 weeks, and 5 rats were sacrificed in each group every other week. a The level of nitric oxide (NO) was detected. b Spinal cord tissues were collected from the PHN group and sham group at 1 w, 2 w and 4 w, immunohistochemical staining was used to detect the level of NO. c Spinal cord tissues were collected from the PHN group and sham group at 1 w, 2 w, and 4 w, respectively, and immunofluorescence staining was performed by using NOS/GFAP. ** P

    Techniques Used: Expressing, Construct, Injection, Immunohistochemistry, Staining, Immunofluorescence

    KCNA2-AS was highly expressed in the spinal cord of PHN model rats, and was correlated with GFAP expression. Varicella zoster virus-infected CV-1 cells (monkey kidney cell line), and 5 × 10 6 cells were injected into rats (PHN group) to construct a PHN rat model. Sham group rats were injected with uninfected CV-1 cells. The experiment lasted 4 weeks, and 5 rats were sacrificed in each group every other week. a Schematic diagram of rat treatment. b Mechanical allodynia was detected by (PWT) before injection or sacrifice in rats. c The mRNA and protein levels of GFAP were detected by qRT-PCR and Western blot. d The expression of five lncRNAs in the spinal cord tissue of rats sacrificed at week 4 was detected by qRT-PCR. e , f The expression levels of KCNA2-AS and H19 were detected by qRT-PCR at different time points. g The correlation between the expression of KCNA2-AS or H19 and GFAP mRNA were analyzed. * P
    Figure Legend Snippet: KCNA2-AS was highly expressed in the spinal cord of PHN model rats, and was correlated with GFAP expression. Varicella zoster virus-infected CV-1 cells (monkey kidney cell line), and 5 × 10 6 cells were injected into rats (PHN group) to construct a PHN rat model. Sham group rats were injected with uninfected CV-1 cells. The experiment lasted 4 weeks, and 5 rats were sacrificed in each group every other week. a Schematic diagram of rat treatment. b Mechanical allodynia was detected by (PWT) before injection or sacrifice in rats. c The mRNA and protein levels of GFAP were detected by qRT-PCR and Western blot. d The expression of five lncRNAs in the spinal cord tissue of rats sacrificed at week 4 was detected by qRT-PCR. e , f The expression levels of KCNA2-AS and H19 were detected by qRT-PCR at different time points. g The correlation between the expression of KCNA2-AS or H19 and GFAP mRNA were analyzed. * P

    Techniques Used: Expressing, Infection, Injection, Construct, Quantitative RT-PCR, Western Blot

    Knockdown of KCNA2-AS in the spinal cord of PHN rats alleviates neuropathic pain. Varicella zoster virus-infected CV-1 cells (monkey kidney cell line), and 5 × 10 6 cells were injected into rats to construct a PHN rat model, and 3 days before, the rats were intrathecal injected (PHN + si-KCNA2-AS group) or no injection (PHN group) of LV-si-KCNA2-AS. The rats were sacrificed after 2 weeks. a Schematic diagram of rat treatment. b Mechanical pain was detected by the PWT method before or 1 and 2 weeks after injection in rats. c The levels of KCNA2-AS were detected in spinal cord tissue by qRT-PCR. d Immunofluorescence staining was performed on the sections of rat spinal cord. e The levels of pSTAT3 and GFAP protein were detected in spinal cord tissue by Western blot. * P
    Figure Legend Snippet: Knockdown of KCNA2-AS in the spinal cord of PHN rats alleviates neuropathic pain. Varicella zoster virus-infected CV-1 cells (monkey kidney cell line), and 5 × 10 6 cells were injected into rats to construct a PHN rat model, and 3 days before, the rats were intrathecal injected (PHN + si-KCNA2-AS group) or no injection (PHN group) of LV-si-KCNA2-AS. The rats were sacrificed after 2 weeks. a Schematic diagram of rat treatment. b Mechanical pain was detected by the PWT method before or 1 and 2 weeks after injection in rats. c The levels of KCNA2-AS were detected in spinal cord tissue by qRT-PCR. d Immunofluorescence staining was performed on the sections of rat spinal cord. e The levels of pSTAT3 and GFAP protein were detected in spinal cord tissue by Western blot. * P

    Techniques Used: Infection, Injection, Construct, Quantitative RT-PCR, Immunofluorescence, Staining, Western Blot

    8) Product Images from "Ajuba receptor mediates the internalization of tumor-secreted GRP78 into macrophages through different endocytosis pathways"

    Article Title: Ajuba receptor mediates the internalization of tumor-secreted GRP78 into macrophages through different endocytosis pathways

    Journal: Oncotarget

    doi: 10.18632/oncotarget.24090

    Phagocytosis is not the only way for secreted GRP78 to enter into macrophages ( A, D, G, J ) Location of GRP78 in FHC, COS-7, DLD1, HeLa cells treated with 40 nM FITC-GRP78 at different time points. Corresponding images were superimposed to determine the location of GRP78. Scale bars represent 6 μm. ( B, E, H, K ) Concentration of GRP78 protein in the above cells. As described in (A, D, G, J), experiments were repeated three times, and 250 cell per time point in each experiment were scored in the quantification analysis using Image J software. ( C, F, I, L ) Levels of internalized protein. As described in (A, D, G, J), Western blot data were superimposed to determine the levels of internalized protein. Mouse anti-GAPDH antibodies was used as a loading control.
    Figure Legend Snippet: Phagocytosis is not the only way for secreted GRP78 to enter into macrophages ( A, D, G, J ) Location of GRP78 in FHC, COS-7, DLD1, HeLa cells treated with 40 nM FITC-GRP78 at different time points. Corresponding images were superimposed to determine the location of GRP78. Scale bars represent 6 μm. ( B, E, H, K ) Concentration of GRP78 protein in the above cells. As described in (A, D, G, J), experiments were repeated three times, and 250 cell per time point in each experiment were scored in the quantification analysis using Image J software. ( C, F, I, L ) Levels of internalized protein. As described in (A, D, G, J), Western blot data were superimposed to determine the levels of internalized protein. Mouse anti-GAPDH antibodies was used as a loading control.

    Techniques Used: Concentration Assay, Software, Western Blot

    9) Product Images from "RNAa Is Conserved in Mammalian Cells"

    Article Title: RNAa Is Conserved in Mammalian Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0008848

    RNA activation of different genes in non-human primate cells. A–G . COS1 and WES cells were transfected with 25 nM of the indicated saRNAs for 3–5 days. Mock samples were transfected in the absence of saRNA. mRNA expression levels were analyzed by real-time PCR and normalized to β-actin. Expression levels were measured as fold induction relative to mock transfections. The results are represented as mean ± SEM of three independent experiments. Species-specific saRNAs were designed to perfectly complement their respective chimpanzee (dsNKX3-1-360-PT) and AGM (dsNKX3-1-360-CA) targeted sequences in WES and COS1 cells, respectively. Statistical significance is indicated (* p
    Figure Legend Snippet: RNA activation of different genes in non-human primate cells. A–G . COS1 and WES cells were transfected with 25 nM of the indicated saRNAs for 3–5 days. Mock samples were transfected in the absence of saRNA. mRNA expression levels were analyzed by real-time PCR and normalized to β-actin. Expression levels were measured as fold induction relative to mock transfections. The results are represented as mean ± SEM of three independent experiments. Species-specific saRNAs were designed to perfectly complement their respective chimpanzee (dsNKX3-1-360-PT) and AGM (dsNKX3-1-360-CA) targeted sequences in WES and COS1 cells, respectively. Statistical significance is indicated (* p

    Techniques Used: Activation Assay, Transfection, Expressing, Real-time Polymerase Chain Reaction

    Activation of p53 by saRNA causes cell cycle arrest and induction of p21 in WES cells. A . WES cells were transfected with 25 nM of the indicated saRNAs for 5 days. Protein levels of p53, PARP and p21 were detected by immunoblot analysis. β-actin served as a loading control. B . WES cells were transfected as in A. Cells were collected, stained with propidium iodide, and processed for analysis by flow cytometry to measure DNA content. Shown are examples of resulting FL2A histograms following analysis with FlowJo software. Cell populations distributed in G0/G1, S, and G2/M phases of the cell cycle are indicated. C . WES cells were transfected with 25 nM of the indicated saRNAs for 72 hrs. p21 mRNA expression was analyzed by real-time PCR. The results represent mean ± SEM of two independent experiments and are plotted as fold induction relative to mock transfections.
    Figure Legend Snippet: Activation of p53 by saRNA causes cell cycle arrest and induction of p21 in WES cells. A . WES cells were transfected with 25 nM of the indicated saRNAs for 5 days. Protein levels of p53, PARP and p21 were detected by immunoblot analysis. β-actin served as a loading control. B . WES cells were transfected as in A. Cells were collected, stained with propidium iodide, and processed for analysis by flow cytometry to measure DNA content. Shown are examples of resulting FL2A histograms following analysis with FlowJo software. Cell populations distributed in G0/G1, S, and G2/M phases of the cell cycle are indicated. C . WES cells were transfected with 25 nM of the indicated saRNAs for 72 hrs. p21 mRNA expression was analyzed by real-time PCR. The results represent mean ± SEM of two independent experiments and are plotted as fold induction relative to mock transfections.

    Techniques Used: Activation Assay, Transfection, Staining, Flow Cytometry, Cytometry, Software, Expressing, Real-time Polymerase Chain Reaction

    10) Product Images from "Suppressors of a Host Range Mutation in the Rabbitpox Virus Serpin SPI-1 Map to Proteins Essential for Viral DNA Replication"

    Article Title: Suppressors of a Host Range Mutation in the Rabbitpox Virus Serpin SPI-1 Map to Proteins Essential for Viral DNA Replication

    Journal:

    doi: 10.1128/JVI.79.14.9168-9179.2005

    Characterization of the RPV sup-2 mutant. (A) RPV DNA synthesis assay. CV-1 cells were infected with wild-type RPV or each designated mutant at an MOI of 10 PFU per cell at 31°C or 41°C. Samples were harvested at intervals up to 24 h postinfection,
    Figure Legend Snippet: Characterization of the RPV sup-2 mutant. (A) RPV DNA synthesis assay. CV-1 cells were infected with wild-type RPV or each designated mutant at an MOI of 10 PFU per cell at 31°C or 41°C. Samples were harvested at intervals up to 24 h postinfection,

    Techniques Used: Mutagenesis, DNA Synthesis, Infection

    Temperature sensitivity of RPV extragenic suppressor mutants. Equivalent dilutions of each RPV isolate were plaqued on monolayers of CV-1 and A549 cells under agarose media in six-well plates at either 37°C or 41°C to assay for temperature
    Figure Legend Snippet: Temperature sensitivity of RPV extragenic suppressor mutants. Equivalent dilutions of each RPV isolate were plaqued on monolayers of CV-1 and A549 cells under agarose media in six-well plates at either 37°C or 41°C to assay for temperature

    Techniques Used:

    Mapping of the sup-2 mutation by marker rescue. The ts property of the RPV sup-2 mutant was used to map the sup-2 mutation by marker rescue. (A and B) Monolayers of CV-1 cells in six-well plates were infected with the RPV sup-2 mutant at an MOI of 0.003
    Figure Legend Snippet: Mapping of the sup-2 mutation by marker rescue. The ts property of the RPV sup-2 mutant was used to map the sup-2 mutation by marker rescue. (A and B) Monolayers of CV-1 cells in six-well plates were infected with the RPV sup-2 mutant at an MOI of 0.003

    Techniques Used: Mutagenesis, Marker, Infection

    RPV host range mutant suppression by sup - 2 does not depend on the hypothetical E ORF E gene. (A) Rescue of the RPV sup-2 mutant at 41°C in CV-1 cells by transfection with wt E9L PCR product replaces the E9ΔH142 allele in the RPV sup-2
    Figure Legend Snippet: RPV host range mutant suppression by sup - 2 does not depend on the hypothetical E ORF E gene. (A) Rescue of the RPV sup-2 mutant at 41°C in CV-1 cells by transfection with wt E9L PCR product replaces the E9ΔH142 allele in the RPV sup-2

    Techniques Used: Mutagenesis, Transfection, Polymerase Chain Reaction

    11) Product Images from "Interaction of porcine reproductive and respiratory syndrome virus proteins with SUMO-conjugating enzyme reveals the SUMOylation of nucleocapsid protein"

    Article Title: Interaction of porcine reproductive and respiratory syndrome virus proteins with SUMO-conjugating enzyme reveals the SUMOylation of nucleocapsid protein

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0189191

    Co-localization of Nsp1β, Nsp4, Nsp9, Nsp10 and N protein with Ubc9. (A) Co-localization of Nsp1β, Nsp4, Nsp9, Nsp10 and N protein with exogenous Ubc9 in HEK293 cells. HEK293 cells were co-transfected with pCMV-HA-Nsp1β, pCMV-HA-Nsp4, pCMV-HA-Nsp9, pCMV-HA-Nsp10 and pCMV-HA-N with pCMV-Myc-Ubc9, respectively. The cells were fixed at 36 h post-transfection and processed by immunostaining with a mouse anti-HA mAb or rabbit anti-Myc PAb and TRITC-conjugated goat anti-mouse IgG or FITC-conjugated goat anti-rabbit IgG, and were then examined by confocal microscopy (600×magnification). Nuclei were stained with DAPI. Co-localization of Nsp1β, Nsp4, Nsp9, Nsp10 and N protein with endogenous Ubc9 in mock-infected MARC-145 cells and PRRSV-infected MARC-145 cells (B) , mock-infected PAMs and PRRSV-infected PAMs (C) . The mock- or PRRSV-infected cells were fixed at 24 h post-infection and processed by immunostaining with a rabbit anti-Ubc9 PAb or mouse anti-Nsp1β, anti-Nsp4, anti-Nsp9, anti-Nsp10 and anti-N mAb, respectively, and then immunostained with TRITC-conjugated goat anti-mouse IgG and FITC-conjugated goat anti-rabbit IgG. Nuclei were stained with DAPI.
    Figure Legend Snippet: Co-localization of Nsp1β, Nsp4, Nsp9, Nsp10 and N protein with Ubc9. (A) Co-localization of Nsp1β, Nsp4, Nsp9, Nsp10 and N protein with exogenous Ubc9 in HEK293 cells. HEK293 cells were co-transfected with pCMV-HA-Nsp1β, pCMV-HA-Nsp4, pCMV-HA-Nsp9, pCMV-HA-Nsp10 and pCMV-HA-N with pCMV-Myc-Ubc9, respectively. The cells were fixed at 36 h post-transfection and processed by immunostaining with a mouse anti-HA mAb or rabbit anti-Myc PAb and TRITC-conjugated goat anti-mouse IgG or FITC-conjugated goat anti-rabbit IgG, and were then examined by confocal microscopy (600×magnification). Nuclei were stained with DAPI. Co-localization of Nsp1β, Nsp4, Nsp9, Nsp10 and N protein with endogenous Ubc9 in mock-infected MARC-145 cells and PRRSV-infected MARC-145 cells (B) , mock-infected PAMs and PRRSV-infected PAMs (C) . The mock- or PRRSV-infected cells were fixed at 24 h post-infection and processed by immunostaining with a rabbit anti-Ubc9 PAb or mouse anti-Nsp1β, anti-Nsp4, anti-Nsp9, anti-Nsp10 and anti-N mAb, respectively, and then immunostained with TRITC-conjugated goat anti-mouse IgG and FITC-conjugated goat anti-rabbit IgG. Nuclei were stained with DAPI.

    Techniques Used: Transfection, Immunostaining, Confocal Microscopy, Staining, Infection

    SUMOylation of PRRSV N protein. (A and B) The expression of Nsp1β, Nsp4, Nsp9, Nsp10 and N proteins in HEK293 cells using a Co-IP assay. HEK293 cells were transfected with pCMV-HA-Nsp1β, pCMV-HA-Nsp4, pCMV-HA-Nsp9, pCMV-HA-Nsp10 and pCMV-HA-N, separately. The cell lysates were immunoprecipitated with an anti-HA mAb and probed with an anti-HA mAb. The left panel indicates the identification of HA-Nsp1β, HA-Nsp4, HA-Nsp9, HA-Nsp10 and HA-N expressed in cell lysates and the right panel shows the Co-IP analyses of HA-Nsp1β, HA-Nsp4, HA-Nsp9, HA-Nsp10 and HA-N from cell lysates. (C) The expression of N and mutated N proteins (C23S and C23A) in HEK293 cells by using a Co-IP assay. HEK293 cells were transfected with pCMV-HA-N, pCMV-HA-N (C23S), pCMV-HA-N (C23A), separately. The cell lysates were immunoprecipitated with an anti-HA mAb and probed with anti-HA mAb. The left panel indicated the identification of HA-N, HA-N (C23S) and HA-N (C23A) expressed in cell lysates and the right panel showed the Co-IP analyses of HA-N, HA-N (C23S) and HA-N (C23A) from cell lysates. (D) The expression of N protein in MARC-145 cells following PRRSV infection using a Co-IP assay. MARC-145 cells were infected with PRRSV JXwn06 at a MOI of 0.1. At 36 h the cell lysates were immunoprecipitated with an anti-N mAb, anti-SUMO1 or anti-SUMO2/3 mAb and probed with these mAb, separately.
    Figure Legend Snippet: SUMOylation of PRRSV N protein. (A and B) The expression of Nsp1β, Nsp4, Nsp9, Nsp10 and N proteins in HEK293 cells using a Co-IP assay. HEK293 cells were transfected with pCMV-HA-Nsp1β, pCMV-HA-Nsp4, pCMV-HA-Nsp9, pCMV-HA-Nsp10 and pCMV-HA-N, separately. The cell lysates were immunoprecipitated with an anti-HA mAb and probed with an anti-HA mAb. The left panel indicates the identification of HA-Nsp1β, HA-Nsp4, HA-Nsp9, HA-Nsp10 and HA-N expressed in cell lysates and the right panel shows the Co-IP analyses of HA-Nsp1β, HA-Nsp4, HA-Nsp9, HA-Nsp10 and HA-N from cell lysates. (C) The expression of N and mutated N proteins (C23S and C23A) in HEK293 cells by using a Co-IP assay. HEK293 cells were transfected with pCMV-HA-N, pCMV-HA-N (C23S), pCMV-HA-N (C23A), separately. The cell lysates were immunoprecipitated with an anti-HA mAb and probed with anti-HA mAb. The left panel indicated the identification of HA-N, HA-N (C23S) and HA-N (C23A) expressed in cell lysates and the right panel showed the Co-IP analyses of HA-N, HA-N (C23S) and HA-N (C23A) from cell lysates. (D) The expression of N protein in MARC-145 cells following PRRSV infection using a Co-IP assay. MARC-145 cells were infected with PRRSV JXwn06 at a MOI of 0.1. At 36 h the cell lysates were immunoprecipitated with an anti-N mAb, anti-SUMO1 or anti-SUMO2/3 mAb and probed with these mAb, separately.

    Techniques Used: Expressing, Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Infection

    Inhibition of PRRSV JXwn06 replication by Ubc9. (A) PRRSV titers in Ubc9-overexpressed MARC-145 cells. MARC-145 cells were transduced with the lentiviruses that were expressing GFP and Ubc9, respectively. The cells were infected with PRRSV JXwn06 at MOI of 0.01 at 24 h post-transduction, and the virus titers were then assayed by a microtitration infectivity assay at the indicated time points post-infection. Data are shown as means ± SD of three independent experiments (** p
    Figure Legend Snippet: Inhibition of PRRSV JXwn06 replication by Ubc9. (A) PRRSV titers in Ubc9-overexpressed MARC-145 cells. MARC-145 cells were transduced with the lentiviruses that were expressing GFP and Ubc9, respectively. The cells were infected with PRRSV JXwn06 at MOI of 0.01 at 24 h post-transduction, and the virus titers were then assayed by a microtitration infectivity assay at the indicated time points post-infection. Data are shown as means ± SD of three independent experiments (** p

    Techniques Used: Inhibition, Transduction, Expressing, Infection

    The interaction of PRRSV Nsp1β, Nsp4, Nsp9, Nsp10 and N protein with Ubc9. (A) The interaction of Nsp1β, Nsp4, Nsp9, Nps10 and N protein with exogenous Ubc9 by using a Co-IP assay. HEK293 cells were co-transfected with the Myc-Ubc9-expressing plasmid and the HA-Nsp1β-, HA-Nsp4-, HA-Nsp9-, HA-Nsp10- and HA-N-expressing plasmid, respectively. The cell lysates were immunoprecipitated with an anti-HA mAb and probed with anti-HA mAb and anti-Myc PAb. The left panel shows the Co-IP analyses of HA-Nsp1β, HA-Nsp4, HA-Nsp9, HA-Nsp10 and HA-N from cell lysates and the right panel indicates the identification of HA-Nsp1β, HA-Nsp4, HA-Nsp9, HA-Nsp10 and HA-N expressed in cell lysates. The asterisk (★) indicates the IgG light chain band with 26 KDa, and the solid triangle (▲) represents the target protein Myc-Ubc9. (B) The interaction of Nsp1β, Nsp4, Nsp9, Nsp10 and N protein with exogenous Ubc9 by using a GST pull-down assay. The cell lysates containing Nsp1β, Nsp4, Nsp9, Nsp10 and N protein individually were pulled down with prokaryotic expressed and purified GST-Ubc9 protein with an anti-GST mAb and probed with anti-HA and anti-GST mAb. (C) The interaction of Nsp1β, Nsp4, Nsp9, Nsp10 and N protein with endogenous Ubc9. MARC-145 cells were transduced with the lentiviruses that were expressing GFP, Nsp1β, Nsp4, Nsp9, Nsp10, or N individually. The cell lysates were immunoprecipitated with an anti-GFP mAb and followed by Western blot analysis with anti-Ubc9 and anti-GFP antibodies. The left panel indicates the identification of GFP, Nsp1β-GFP, Nsp4-GFP, Nsp9-GFP, Nsp10-GFP and N-GFP expressed in cell lysates, while the right panel shows the Co-IP analyses of GFP, Nsp1β-GFP, Nsp4-GFP, Nsp9-GFP, Nsp10-GFP and N-GFP from cell lysates.
    Figure Legend Snippet: The interaction of PRRSV Nsp1β, Nsp4, Nsp9, Nsp10 and N protein with Ubc9. (A) The interaction of Nsp1β, Nsp4, Nsp9, Nps10 and N protein with exogenous Ubc9 by using a Co-IP assay. HEK293 cells were co-transfected with the Myc-Ubc9-expressing plasmid and the HA-Nsp1β-, HA-Nsp4-, HA-Nsp9-, HA-Nsp10- and HA-N-expressing plasmid, respectively. The cell lysates were immunoprecipitated with an anti-HA mAb and probed with anti-HA mAb and anti-Myc PAb. The left panel shows the Co-IP analyses of HA-Nsp1β, HA-Nsp4, HA-Nsp9, HA-Nsp10 and HA-N from cell lysates and the right panel indicates the identification of HA-Nsp1β, HA-Nsp4, HA-Nsp9, HA-Nsp10 and HA-N expressed in cell lysates. The asterisk (★) indicates the IgG light chain band with 26 KDa, and the solid triangle (▲) represents the target protein Myc-Ubc9. (B) The interaction of Nsp1β, Nsp4, Nsp9, Nsp10 and N protein with exogenous Ubc9 by using a GST pull-down assay. The cell lysates containing Nsp1β, Nsp4, Nsp9, Nsp10 and N protein individually were pulled down with prokaryotic expressed and purified GST-Ubc9 protein with an anti-GST mAb and probed with anti-HA and anti-GST mAb. (C) The interaction of Nsp1β, Nsp4, Nsp9, Nsp10 and N protein with endogenous Ubc9. MARC-145 cells were transduced with the lentiviruses that were expressing GFP, Nsp1β, Nsp4, Nsp9, Nsp10, or N individually. The cell lysates were immunoprecipitated with an anti-GFP mAb and followed by Western blot analysis with anti-Ubc9 and anti-GFP antibodies. The left panel indicates the identification of GFP, Nsp1β-GFP, Nsp4-GFP, Nsp9-GFP, Nsp10-GFP and N-GFP expressed in cell lysates, while the right panel shows the Co-IP analyses of GFP, Nsp1β-GFP, Nsp4-GFP, Nsp9-GFP, Nsp10-GFP and N-GFP from cell lysates.

    Techniques Used: Co-Immunoprecipitation Assay, Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Pull Down Assay, Purification, Transduction, Western Blot

    12) Product Images from "Preclinical Testing Oncolytic Vaccinia Virus Strain GLV-5b451 Expressing an Anti-VEGF Single-Chain Antibody for Canine Cancer Therapy"

    Article Title: Preclinical Testing Oncolytic Vaccinia Virus Strain GLV-5b451 Expressing an Anti-VEGF Single-Chain Antibody for Canine Cancer Therapy

    Journal: Viruses

    doi: 10.3390/v7072811

    Replication capacity of the vaccinia virus strains LIVP 6.1.1 ( A ) and GLV-5b451 ( B ) in different canine cancer cell lines. For the viral replication assay, MTH52c, ZMTH3, CT1258 or STSA-1 cells grown in 24-well plates were infected with either LIVP 6.1.1 or GLV-5b451 at an MOI of 0.1. Cells and supernatants were collected for the determination of virus titers at various time points. Viral titers were determined as pfu per ml in triplicates by standard plaque assay in CV-1 cell monolayers. Averages plus standard deviation are plotted. The data represent three independent experiments.
    Figure Legend Snippet: Replication capacity of the vaccinia virus strains LIVP 6.1.1 ( A ) and GLV-5b451 ( B ) in different canine cancer cell lines. For the viral replication assay, MTH52c, ZMTH3, CT1258 or STSA-1 cells grown in 24-well plates were infected with either LIVP 6.1.1 or GLV-5b451 at an MOI of 0.1. Cells and supernatants were collected for the determination of virus titers at various time points. Viral titers were determined as pfu per ml in triplicates by standard plaque assay in CV-1 cell monolayers. Averages plus standard deviation are plotted. The data represent three independent experiments.

    Techniques Used: Viral Replication Assay, Infection, Plaque Assay, Standard Deviation

    13) Product Images from "Replication efficiency of oncolytic vaccinia virus in cell cultures prognosticates the virulence and antitumor efficacy in mice"

    Article Title: Replication efficiency of oncolytic vaccinia virus in cell cultures prognosticates the virulence and antitumor efficacy in mice

    Journal: Journal of Translational Medicine

    doi: 10.1186/1479-5876-9-164

    Excision of the foreign expression cassettes from GLV-1h68 enhances virus replication efficiency . (A) Viral growth curves. GI-101A cells were infected with GLV-1h68 or its derivatives at an MOI of 0.01 or 10, and harvested at 24, 48, and 72 hpi. Viral titers were determined in CV-1 cells. The values are the mean of triplicate samples, and the bars indicate SD. The data represents two independent experiments. Statistical analysis was performed using two-way ANOVA. *, **, and *** indicate P
    Figure Legend Snippet: Excision of the foreign expression cassettes from GLV-1h68 enhances virus replication efficiency . (A) Viral growth curves. GI-101A cells were infected with GLV-1h68 or its derivatives at an MOI of 0.01 or 10, and harvested at 24, 48, and 72 hpi. Viral titers were determined in CV-1 cells. The values are the mean of triplicate samples, and the bars indicate SD. The data represents two independent experiments. Statistical analysis was performed using two-way ANOVA. *, **, and *** indicate P

    Techniques Used: Expressing, Infection

    GLV-1h68 and its derivatives . (A) Schematic representation of the genomic structures of the recombinant vaccinia virus GLV-1h68 and its marker gene expression cassette removal derivatives. PE/L, P11, and P7.5 are VACV synthetic early/late, 11K, and 7.5K promoters, respectively. TfR is human transferin receptor inserted in the reverse orientation with respect to the promoter PE/L. (B) Marker gene expression and genotype verification. CV-1 cells were infected with each individual virus strain. Two days post-infection, the GFP expression was visualized by fluorescence microscopy and expression of β-galactosidase and β-glucuronidase was detected by X-gal and X-GLcA staining, respectively.
    Figure Legend Snippet: GLV-1h68 and its derivatives . (A) Schematic representation of the genomic structures of the recombinant vaccinia virus GLV-1h68 and its marker gene expression cassette removal derivatives. PE/L, P11, and P7.5 are VACV synthetic early/late, 11K, and 7.5K promoters, respectively. TfR is human transferin receptor inserted in the reverse orientation with respect to the promoter PE/L. (B) Marker gene expression and genotype verification. CV-1 cells were infected with each individual virus strain. Two days post-infection, the GFP expression was visualized by fluorescence microscopy and expression of β-galactosidase and β-glucuronidase was detected by X-gal and X-GLcA staining, respectively.

    Techniques Used: Recombinant, Marker, Expressing, Infection, Fluorescence, Microscopy, Staining

    14) Product Images from "Variable Sensitivity to Substitutions in the N-Terminal Heptad Repeat of Mason-Pfizer Monkey Virus Transmembrane Protein"

    Article Title: Variable Sensitivity to Substitutions in the N-Terminal Heptad Repeat of Mason-Pfizer Monkey Virus Transmembrane Protein

    Journal: Journal of Virology

    doi: 10.1128/JVI.77.14.7779-7785.2003

    Synthesis and processing of mutant and wild-type (WT) glycoproteins. (A) Diagram of the M-PMV TM protein showing the location of the HR1 domain of M-PMV relative to the rest of the glycoprotein. The positions of the primers used to introduce mutations are shown. (B) COS-1 cells were transfected with the pTMT expression vector containing either wild-type or mutant env genes. At 48 h posttransfection, the cells were labeled with [ 3 H]leucine for 30 min and immunoprecipitated with goat anti-M-PMV serum as described in the text. The mutant designations are shown above each lane, and the position of the precursor glycoprotein, Pr86, is indicated to the left. Mock, mock transfected. (C) Processing of the env gene polyprotein precursor protein. Following a 4-h chase in unlabeled medium, Env proteins were immunoprecipitated with goat anti-M-PMV serum and electrophoresed in SDS-PAGE. The positions of the precursor glycoprotein, Pr86, and the cleavage products, gp70 and gp22, are shown. (D) Position of gp22 after longer exposure. (E) Secretion of SU into culture media. Following pulse-chase, the culture media were collected, immunoprecipitated with goat anti-M-PMV serum, and analyzed for the released SU proteins.
    Figure Legend Snippet: Synthesis and processing of mutant and wild-type (WT) glycoproteins. (A) Diagram of the M-PMV TM protein showing the location of the HR1 domain of M-PMV relative to the rest of the glycoprotein. The positions of the primers used to introduce mutations are shown. (B) COS-1 cells were transfected with the pTMT expression vector containing either wild-type or mutant env genes. At 48 h posttransfection, the cells were labeled with [ 3 H]leucine for 30 min and immunoprecipitated with goat anti-M-PMV serum as described in the text. The mutant designations are shown above each lane, and the position of the precursor glycoprotein, Pr86, is indicated to the left. Mock, mock transfected. (C) Processing of the env gene polyprotein precursor protein. Following a 4-h chase in unlabeled medium, Env proteins were immunoprecipitated with goat anti-M-PMV serum and electrophoresed in SDS-PAGE. The positions of the precursor glycoprotein, Pr86, and the cleavage products, gp70 and gp22, are shown. (D) Position of gp22 after longer exposure. (E) Secretion of SU into culture media. Following pulse-chase, the culture media were collected, immunoprecipitated with goat anti-M-PMV serum, and analyzed for the released SU proteins.

    Techniques Used: Mutagenesis, Introduce, Transfection, Expressing, Plasmid Preparation, Labeling, Immunoprecipitation, SDS Page, Pulse Chase

    Cell-cell fusion assay of envelope glycoproteins expressed from the pTMT expression vector. COS-1 cells were transfected with the pTMT expression vector containing either the mutant or wild-type (WT) env gene. COS-1 cells expressing glycoproteins were mixed 1:2 with GHOST cells, replated, and analyzed as described in Materials and Methods. (A) Cells were gated on size and then analyzed for expression of GFP by quantitating excitation at 488 nm. (B) The percentage of fused GFP-positive cells was calculated for each mutant and normalized to the wild type as shown. The graph depicts the mean of three independent experiments (± standard deviation). The mutant designation is shown below each histogram.
    Figure Legend Snippet: Cell-cell fusion assay of envelope glycoproteins expressed from the pTMT expression vector. COS-1 cells were transfected with the pTMT expression vector containing either the mutant or wild-type (WT) env gene. COS-1 cells expressing glycoproteins were mixed 1:2 with GHOST cells, replated, and analyzed as described in Materials and Methods. (A) Cells were gated on size and then analyzed for expression of GFP by quantitating excitation at 488 nm. (B) The percentage of fused GFP-positive cells was calculated for each mutant and normalized to the wild type as shown. The graph depicts the mean of three independent experiments (± standard deviation). The mutant designation is shown below each histogram.

    Techniques Used: Cell-Cell Fusion Assay, Expressing, Plasmid Preparation, Transfection, Mutagenesis, Standard Deviation

    Expression of envelope glycoprotein mutants in the context of provirus. The mutant env genes in pSARM4 were transfected into COS-1 cells and metabolically labeled as described in Materials and Methods. (A) Cell lysates were immunoprecipitated with goat anti-M-PMV serum and analyzed in SDS-PAGE. The mutant designation is shown above each lane, and the positions of the viral bands are indicated on the left. WT, wild type; Mock, mock transfected. (B and C) Incorporation of mutant glycoprotein into virions. Virus-containing supernatants from metabolically labeled COS-1 cells transfected with either wild-type or mutant pSARM4 constructs were centrifuged through a 25% sucrose cushion. The virus pellets were recovered and immunoprecipitated with goat anti-M-PMV serum (B) or electrophoresed directly (C). The positions of the viral bands are indicated on the left.
    Figure Legend Snippet: Expression of envelope glycoprotein mutants in the context of provirus. The mutant env genes in pSARM4 were transfected into COS-1 cells and metabolically labeled as described in Materials and Methods. (A) Cell lysates were immunoprecipitated with goat anti-M-PMV serum and analyzed in SDS-PAGE. The mutant designation is shown above each lane, and the positions of the viral bands are indicated on the left. WT, wild type; Mock, mock transfected. (B and C) Incorporation of mutant glycoprotein into virions. Virus-containing supernatants from metabolically labeled COS-1 cells transfected with either wild-type or mutant pSARM4 constructs were centrifuged through a 25% sucrose cushion. The virus pellets were recovered and immunoprecipitated with goat anti-M-PMV serum (B) or electrophoresed directly (C). The positions of the viral bands are indicated on the left.

    Techniques Used: Expressing, Mutagenesis, Transfection, Metabolic Labelling, Labeling, Immunoprecipitation, SDS Page, Construct

    Single-round infectivity assay of virus. COS-1 cells were cotransfected with pTMT and pMΤΔΕ expression vectors as described in Materials and Methods. Culture medium from cells expressing virus was filtered and normalized for RT activity. The normalized medium was used to infect GHOST cells. (A) Cells were gated on size and granularity and then analyzed for GFP expression. WT, wild type. (B) A total of 3 × 10 4 cells were analyzed for each construct, and the number of GFP-expressing cells relative to the wild type was plotted. The graph shows the mean from three independent experiments (± standard deviation).
    Figure Legend Snippet: Single-round infectivity assay of virus. COS-1 cells were cotransfected with pTMT and pMΤΔΕ expression vectors as described in Materials and Methods. Culture medium from cells expressing virus was filtered and normalized for RT activity. The normalized medium was used to infect GHOST cells. (A) Cells were gated on size and granularity and then analyzed for GFP expression. WT, wild type. (B) A total of 3 × 10 4 cells were analyzed for each construct, and the number of GFP-expressing cells relative to the wild type was plotted. The graph shows the mean from three independent experiments (± standard deviation).

    Techniques Used: Infection, Expressing, Activity Assay, Construct, Standard Deviation

    15) Product Images from "Dynamic transcriptome profiling dataset of vaccinia virus obtained from long-read sequencing techniques"

    Article Title: Dynamic transcriptome profiling dataset of vaccinia virus obtained from long-read sequencing techniques

    Journal: GigaScience

    doi: 10.1093/gigascience/giy139

    Comparison of the read lengths mapped to the host genome of this and other studies. It must be noted here that the analyzed cell lines are from different organisms and/or they were infected with different viruses using different incubation time points. (A-C ) RSII and Sequel platforms provide relatively fixed read length (RSII: 800–1,400 bp; Sequel: 1,050–1,500 bp). The average read length of CV-1 samples is longer than those of the MRC-5 in the RSII PolyA-sequencing; however, the opposite result has been obtained with the random-primed RSII and the Sequel PolyA-Seq. ( D-G) The MinION platform produces greater length variance (250–1,200 bp) except for the Cap-Seq approach, which shows a very small difference between the read lengths among the four different cell lines. Cell lines: African green monkey kidney fibroblast cells (CV-1) infected with VACV or herpes simplex virus type 1 (HSV-1); human lung fibroblast cells (MRC-5) infected with human cytomegalovirus (HCMV) or varicella-zoster virus (VZV); porcine kidney 15 (PK-15) cell line infected with pseudorabies virus (PRV); and Sf9 insect cell line infected with the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV).
    Figure Legend Snippet: Comparison of the read lengths mapped to the host genome of this and other studies. It must be noted here that the analyzed cell lines are from different organisms and/or they were infected with different viruses using different incubation time points. (A-C ) RSII and Sequel platforms provide relatively fixed read length (RSII: 800–1,400 bp; Sequel: 1,050–1,500 bp). The average read length of CV-1 samples is longer than those of the MRC-5 in the RSII PolyA-sequencing; however, the opposite result has been obtained with the random-primed RSII and the Sequel PolyA-Seq. ( D-G) The MinION platform produces greater length variance (250–1,200 bp) except for the Cap-Seq approach, which shows a very small difference between the read lengths among the four different cell lines. Cell lines: African green monkey kidney fibroblast cells (CV-1) infected with VACV or herpes simplex virus type 1 (HSV-1); human lung fibroblast cells (MRC-5) infected with human cytomegalovirus (HCMV) or varicella-zoster virus (VZV); porcine kidney 15 (PK-15) cell line infected with pseudorabies virus (PRV); and Sf9 insect cell line infected with the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV).

    Techniques Used: Infection, Incubation, Sequencing, Random Primed

    16) Product Images from "Reprogramming antitumor immunity against chemoresistant ovarian cancer by a CXCR4 antagonist-armed viral oncotherapy"

    Article Title: Reprogramming antitumor immunity against chemoresistant ovarian cancer by a CXCR4 antagonist-armed viral oncotherapy

    Journal: Molecular Therapy Oncolytics

    doi: 10.1038/mto.2016.34

    Phenotypic characterization of ID8-R and CAOV2-R ovarian tumor cells and their parental counterparts. Flow cytometry analysis of CD44 ( a ) and CXCR4 ( b ) expression in parental and drug-resistant variants was performed on single-cell suspensions with specific mAbs. Background staining was assessed using isotype control Abs. Data are from one representative experiment of three performed. ( c ) Susceptibility of ID8-R and CAOV2-R to vaccinia virus infection. The parental and drug-resistant tumor cells were cultured as a monolayer before infection with OVV-EGFP (MOI = 1). The expression of EGFP in infected cells was examined under an immunofluorescence microscope 24 hours later. Scale bars = 25 µm. One representative experiment of three performed is shown. ( d ) The number of EGFP-expressing cells in each culture was determined by examining single-cell suspensions 24 hours after infection by flow cytometry analysis. Background staining depicts uninfected controls. One representative experiment of four independent experiments performed is shown. ( e ) Replication of OVV-EGFP in different cultures was determined by titrating viral particles released from the infected cells at different time points by plaque assays in CV-1 cell monolayers. Results are presented as the mean of plaque forming units (PFU)/million cells ± SD of three independent experiments performed in duplicate. * P
    Figure Legend Snippet: Phenotypic characterization of ID8-R and CAOV2-R ovarian tumor cells and their parental counterparts. Flow cytometry analysis of CD44 ( a ) and CXCR4 ( b ) expression in parental and drug-resistant variants was performed on single-cell suspensions with specific mAbs. Background staining was assessed using isotype control Abs. Data are from one representative experiment of three performed. ( c ) Susceptibility of ID8-R and CAOV2-R to vaccinia virus infection. The parental and drug-resistant tumor cells were cultured as a monolayer before infection with OVV-EGFP (MOI = 1). The expression of EGFP in infected cells was examined under an immunofluorescence microscope 24 hours later. Scale bars = 25 µm. One representative experiment of three performed is shown. ( d ) The number of EGFP-expressing cells in each culture was determined by examining single-cell suspensions 24 hours after infection by flow cytometry analysis. Background staining depicts uninfected controls. One representative experiment of four independent experiments performed is shown. ( e ) Replication of OVV-EGFP in different cultures was determined by titrating viral particles released from the infected cells at different time points by plaque assays in CV-1 cell monolayers. Results are presented as the mean of plaque forming units (PFU)/million cells ± SD of three independent experiments performed in duplicate. * P

    Techniques Used: Flow Cytometry, Cytometry, Expressing, Staining, Infection, Cell Culture, Immunofluorescence, Microscopy

    17) Product Images from "The adenomatous polyposis coli-binding protein EB1 is associated with cytoplasmic and spindle microtubules"

    Article Title: The adenomatous polyposis coli-binding protein EB1 is associated with cytoplasmic and spindle microtubules

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

    doi:

    Colocalization of EB1 with cytoplasmic microtubules and the centrosome. Unsynchronized CV-1 cells were fixed in methanol and processed for dual immunofluorescence with antibodies directed against EB1 (GD10) and α-tubulin (YL1/2), in the presence or absence of the microtubule destabilizing drug nocodazole (10 μM). ( a , a ′, and a ′′) Control untreated cells. ( b , b ′, and b ′′) Cells treated with nocodazole for 1 hr and fixed in methanol at −20°C. ( c , c ′, and c ′′) Cells fixed in methanol 5 min after nocodazole removal. Staining was analyzed by confocal microscopy. (Bar, 10 μm.)
    Figure Legend Snippet: Colocalization of EB1 with cytoplasmic microtubules and the centrosome. Unsynchronized CV-1 cells were fixed in methanol and processed for dual immunofluorescence with antibodies directed against EB1 (GD10) and α-tubulin (YL1/2), in the presence or absence of the microtubule destabilizing drug nocodazole (10 μM). ( a , a ′, and a ′′) Control untreated cells. ( b , b ′, and b ′′) Cells treated with nocodazole for 1 hr and fixed in methanol at −20°C. ( c , c ′, and c ′′) Cells fixed in methanol 5 min after nocodazole removal. Staining was analyzed by confocal microscopy. (Bar, 10 μm.)

    Techniques Used: Immunofluorescence, Staining, Confocal Microscopy

    Subcellular localization of EB1 in mammalian cells. ( a ) Specificity of the anti-EB1 mAbs GD10 and EA3 for EB1 in cell lysates (lanes 1 and 3) and immunoprecipitation (lanes 2 and 4). Cell lysates and immunoprecipitated proteins from CV-1 cells, prepared as described, were separated in 10% SDS-polyacrylamide gels, transferred to poly(vinylidene difluoride) membranes, probed with anti-EB1 antibodies (GD10 and EA3), and detected by ECL; heavy chain (HC) and light chain (LC) are indicated. Interphase CV-1 ( b and c ) and SW480 cells ( d ) were stained with GD10 antibody after methanol fixation at −20°C. A secondary donkey anti-mouse rhodamine-conjugated antibody was used, and the samples were analyzed by confocal microscopy. [Bars: 25 μm ( b ) and 10 μm ( c and d ).]
    Figure Legend Snippet: Subcellular localization of EB1 in mammalian cells. ( a ) Specificity of the anti-EB1 mAbs GD10 and EA3 for EB1 in cell lysates (lanes 1 and 3) and immunoprecipitation (lanes 2 and 4). Cell lysates and immunoprecipitated proteins from CV-1 cells, prepared as described, were separated in 10% SDS-polyacrylamide gels, transferred to poly(vinylidene difluoride) membranes, probed with anti-EB1 antibodies (GD10 and EA3), and detected by ECL; heavy chain (HC) and light chain (LC) are indicated. Interphase CV-1 ( b and c ) and SW480 cells ( d ) were stained with GD10 antibody after methanol fixation at −20°C. A secondary donkey anti-mouse rhodamine-conjugated antibody was used, and the samples were analyzed by confocal microscopy. [Bars: 25 μm ( b ) and 10 μm ( c and d ).]

    Techniques Used: Immunoprecipitation, Staining, Confocal Microscopy

    EB1 localizes to the mitotic apparatus during cell division. Unsynchronized CV-1 cells were fixed in methanol and stained with the anti-EB1 antibody GD10 and a secondary donkey anti-mouse rhodamine-conjugated antibody. DNA was counterstained with 4,6-diamidino-2-phenylindole (DAPI). ( a , a ′, and a ′′) Prophase. ( b , b ′, and b ′′) Prometaphase. ( c , c ′, and c ′′) Metaphase. ( d , d ′, and d ′′) Anaphase. ( e , e ′, and e ′′) Telophase. ( f , f ′, and f ′′) Cytokinesis.
    Figure Legend Snippet: EB1 localizes to the mitotic apparatus during cell division. Unsynchronized CV-1 cells were fixed in methanol and stained with the anti-EB1 antibody GD10 and a secondary donkey anti-mouse rhodamine-conjugated antibody. DNA was counterstained with 4,6-diamidino-2-phenylindole (DAPI). ( a , a ′, and a ′′) Prophase. ( b , b ′, and b ′′) Prometaphase. ( c , c ′, and c ′′) Metaphase. ( d , d ′, and d ′′) Anaphase. ( e , e ′, and e ′′) Telophase. ( f , f ′, and f ′′) Cytokinesis.

    Techniques Used: Staining

    18) Product Images from "Insertion of the human sodium iodide symporter to facilitate deep tissue imaging does not alter oncolytic or replication capability of a novel vaccinia virus"

    Article Title: Insertion of the human sodium iodide symporter to facilitate deep tissue imaging does not alter oncolytic or replication capability of a novel vaccinia virus

    Journal: Journal of Translational Medicine

    doi: 10.1186/1479-5876-9-36

    GLV-1h153 construct . a. GLV-1h153 was derived from GLV-1h68 by replacing the gus A expression cassette at the A56R locus with the hNIS expression cassette through in vivo homologous recombination. Both viruses contain RUC-GFP and lacZ expression cassettes at the F14.5L and J2R loci, respectively. PE, PE/L, P11, and P7.5 are VACV synthetic early, synthetic early/late, 11K, and 7.5K promoters, respectively. TFR is human transferrin receptor inserted in the reverse orientation with respect to the promoter PE/L.b. Confirmation of GFP , LacZ , and lack of gus A marker gene expression in GLV-1h153 infected CV-1 cells. While the gus A gene cassette is expressed in cells infected with parent virus GLV-1h68, this has been replaced by the hNIS gene cassette in GLV-1h153, leading to loss of gus A expression.
    Figure Legend Snippet: GLV-1h153 construct . a. GLV-1h153 was derived from GLV-1h68 by replacing the gus A expression cassette at the A56R locus with the hNIS expression cassette through in vivo homologous recombination. Both viruses contain RUC-GFP and lacZ expression cassettes at the F14.5L and J2R loci, respectively. PE, PE/L, P11, and P7.5 are VACV synthetic early, synthetic early/late, 11K, and 7.5K promoters, respectively. TFR is human transferrin receptor inserted in the reverse orientation with respect to the promoter PE/L.b. Confirmation of GFP , LacZ , and lack of gus A marker gene expression in GLV-1h153 infected CV-1 cells. While the gus A gene cassette is expressed in cells infected with parent virus GLV-1h68, this has been replaced by the hNIS gene cassette in GLV-1h153, leading to loss of gus A expression.

    Techniques Used: Construct, Derivative Assay, Expressing, In Vivo, Homologous Recombination, Marker, Infection

    19) Product Images from "Aerosol SARS-CoV-2 in hospitals and long-term care homes during the COVID-19 pandemic"

    Article Title: Aerosol SARS-CoV-2 in hospitals and long-term care homes during the COVID-19 pandemic

    Journal: medRxiv

    doi: 10.1101/2021.05.31.21257841

    Impact of air sampling on SARS-CoV-2 recovery from gelatin filters. Inocula (A), (B) and (C) were intended to consist of approximately 3.5, 2.5, and 1.5 Log TCID50 of SARS-CoV-2/mL, respectively, with (A) undiluted (neat), and (B) and (C) diluted from the initial Liquid Inoculum. Following one hour of drying, inoculated filters were either placed in UPAS units for 16 hours (Air Sampler), dissolved in VTM and processed immediately (Control), or dissolved at placed at 4°C overnight prior to processing (4°C). Results represent viable virus recovered in VeroE6 cells by TCID 50 assay. Whiskers represent standard deviation of replicate samples.
    Figure Legend Snippet: Impact of air sampling on SARS-CoV-2 recovery from gelatin filters. Inocula (A), (B) and (C) were intended to consist of approximately 3.5, 2.5, and 1.5 Log TCID50 of SARS-CoV-2/mL, respectively, with (A) undiluted (neat), and (B) and (C) diluted from the initial Liquid Inoculum. Following one hour of drying, inoculated filters were either placed in UPAS units for 16 hours (Air Sampler), dissolved in VTM and processed immediately (Control), or dissolved at placed at 4°C overnight prior to processing (4°C). Results represent viable virus recovered in VeroE6 cells by TCID 50 assay. Whiskers represent standard deviation of replicate samples.

    Techniques Used: Sampling, Standard Deviation

    20) Product Images from "Doxycycline inhibition of a pseudotyped virus transduction does not translate to inhibition of SARS-CoV-2 infectivity"

    Article Title: Doxycycline inhibition of a pseudotyped virus transduction does not translate to inhibition of SARS-CoV-2 infectivity

    Journal: bioRxiv

    doi: 10.1101/2021.07.30.454436

    Doxycycline did not inhibit SARS-CoV-2 authentic virus replication. Vero E6 cells were pretreated for 4 h with doxycycline or gentamicin (100 μM), then infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.01 for 1 h at 37°C with doxycycline or gentamicin (100 μM). Cells were then washed and cultured for 48 h in fresh medium containing doxycycline or gentamicin (100 μM). Non-infected cells (NI) or cells infected without doxycycline or gentamicin treatment (SARS-CoV-2) were used as controls. ( A ) Cells were imaged with an optical microscope to detect typical SARS-CoV-2-induced cytolytic effects (original magnification 10X). ( B ) Viral yield was quantified in the cell supernatant by qRT-PCR. ( C ) Quantification of SARS-CoV-2 genomes at the intracellular level by qRT-PCR. Data are the mean ± SD of at least three independent replicates.
    Figure Legend Snippet: Doxycycline did not inhibit SARS-CoV-2 authentic virus replication. Vero E6 cells were pretreated for 4 h with doxycycline or gentamicin (100 μM), then infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.01 for 1 h at 37°C with doxycycline or gentamicin (100 μM). Cells were then washed and cultured for 48 h in fresh medium containing doxycycline or gentamicin (100 μM). Non-infected cells (NI) or cells infected without doxycycline or gentamicin treatment (SARS-CoV-2) were used as controls. ( A ) Cells were imaged with an optical microscope to detect typical SARS-CoV-2-induced cytolytic effects (original magnification 10X). ( B ) Viral yield was quantified in the cell supernatant by qRT-PCR. ( C ) Quantification of SARS-CoV-2 genomes at the intracellular level by qRT-PCR. Data are the mean ± SD of at least three independent replicates.

    Techniques Used: Infection, Cell Culture, Microscopy, Quantitative RT-PCR

    Doxycycline inhibited the transduction of pseudotyped retroviral vector exposing the SARS-CoV-2 S protein. ( A ) Representive image of two isolated pseudotyped retrovirus particles exposing the SARS-CoV-2 S protein. CA, capsid; EN, envelope; MA, matrix; spikes are indicated by red arrowheads. Scale bar 50 nm. Dose-response effect of doxycycline in ( B ) VeroE6 and ( C ) HEK293-ACE2 cells. The y-axis showed the mean± SD percentage of GFP-transduced cells in relation to control cells. The top limit was set as the average-vehicle only control percentage of this assay. Effects of ( D ) 1 or 100 µM doxycycline and gentamicin 1 or 100 µM ( E ) on transduction of the pseudotyped retroviral vector with SARS-CoV-2 S protein in Vero E6 and HEK293-ACE cells. Data are the mean± SD of the percentage of GFP-transduced cells in relation to control cells transduced with vehicle only (dotted line). The percentage of Vero E6 cells transduced with the retroviral vector without SARS-CoV-2 S protein (No-Spike) is reported as negative control. ****p
    Figure Legend Snippet: Doxycycline inhibited the transduction of pseudotyped retroviral vector exposing the SARS-CoV-2 S protein. ( A ) Representive image of two isolated pseudotyped retrovirus particles exposing the SARS-CoV-2 S protein. CA, capsid; EN, envelope; MA, matrix; spikes are indicated by red arrowheads. Scale bar 50 nm. Dose-response effect of doxycycline in ( B ) VeroE6 and ( C ) HEK293-ACE2 cells. The y-axis showed the mean± SD percentage of GFP-transduced cells in relation to control cells. The top limit was set as the average-vehicle only control percentage of this assay. Effects of ( D ) 1 or 100 µM doxycycline and gentamicin 1 or 100 µM ( E ) on transduction of the pseudotyped retroviral vector with SARS-CoV-2 S protein in Vero E6 and HEK293-ACE cells. Data are the mean± SD of the percentage of GFP-transduced cells in relation to control cells transduced with vehicle only (dotted line). The percentage of Vero E6 cells transduced with the retroviral vector without SARS-CoV-2 S protein (No-Spike) is reported as negative control. ****p

    Techniques Used: Transduction, Plasmid Preparation, Isolation, Negative Control

    Dose-response effect of gentamicin in VeroE6 cells. The y-axis showed the mean± SD percentage of GFP-transduced cells in relation to control cells treated with vehicle alone (Vhc). The percentage of Vero E6 cells transduced with the retroviral vector without SARS-CoV-2 S protein (No-Spike) is reported as negative control.
    Figure Legend Snippet: Dose-response effect of gentamicin in VeroE6 cells. The y-axis showed the mean± SD percentage of GFP-transduced cells in relation to control cells treated with vehicle alone (Vhc). The percentage of Vero E6 cells transduced with the retroviral vector without SARS-CoV-2 S protein (No-Spike) is reported as negative control.

    Techniques Used: Transduction, Plasmid Preparation, Negative Control

    21) Product Images from "Quinacrine, an Antimalarial Drug with Strong Activity Inhibiting SARS-CoV-2 Viral Replication In Vitro"

    Article Title: Quinacrine, an Antimalarial Drug with Strong Activity Inhibiting SARS-CoV-2 Viral Replication In Vitro

    Journal: Viruses

    doi: 10.3390/v13010121

    Inhibition/cytotoxicity of quinacrine (Qx) and chloroquine (CQ) in Vero E6 cells. Vero E6 cells were incubated in presence of different concentrations of Qx ( A , B , blue line) or CQ ( C and D , blue line) and 48 h later the cytotoxicity was evaluated by MTT. Additionally, Vero E6 cells were incubate with SARS-CoV-2 at multiplicities of infections (MOIs) of 0.1 ( A , C ) and 0.01 ( B ) at the same time of Qx and CQ (black lines). Data represent the mean ± SD from at least three independent experiments.
    Figure Legend Snippet: Inhibition/cytotoxicity of quinacrine (Qx) and chloroquine (CQ) in Vero E6 cells. Vero E6 cells were incubated in presence of different concentrations of Qx ( A , B , blue line) or CQ ( C and D , blue line) and 48 h later the cytotoxicity was evaluated by MTT. Additionally, Vero E6 cells were incubate with SARS-CoV-2 at multiplicities of infections (MOIs) of 0.1 ( A , C ) and 0.01 ( B ) at the same time of Qx and CQ (black lines). Data represent the mean ± SD from at least three independent experiments.

    Techniques Used: Inhibition, Incubation, MTT Assay

    Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) characterization in Vero E6 cells. ( A ) Viral infection of Vero E6 cells. Non-infected Vero E6 cells and Vero E6 cells, inoculated with 500 μL of viral stock, were incubated for 24 h at 37 °C, and afterwards visualized by electron microscopy. Viral particles are attached to cell membrane and endocytosed. Endocytosed viral particles (red) are internalized into early endosomes and late endosomes. Under electron microscope, viral particles with morphology similar to coronavirus 2019 are observed. Approximately 63 nm in diameter (red bar) and spike protein was observed. ( B ) Molecular identification. Isolated viruses were identified by real time RT-PCR from the viral RNA using specific probes amplifying ORF1ab gene (FAM) and N gene (VIC).
    Figure Legend Snippet: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) characterization in Vero E6 cells. ( A ) Viral infection of Vero E6 cells. Non-infected Vero E6 cells and Vero E6 cells, inoculated with 500 μL of viral stock, were incubated for 24 h at 37 °C, and afterwards visualized by electron microscopy. Viral particles are attached to cell membrane and endocytosed. Endocytosed viral particles (red) are internalized into early endosomes and late endosomes. Under electron microscope, viral particles with morphology similar to coronavirus 2019 are observed. Approximately 63 nm in diameter (red bar) and spike protein was observed. ( B ) Molecular identification. Isolated viruses were identified by real time RT-PCR from the viral RNA using specific probes amplifying ORF1ab gene (FAM) and N gene (VIC).

    Techniques Used: Infection, Incubation, Electron Microscopy, Microscopy, Isolation, Quantitative RT-PCR

    Effect of Qx against SARS-CoV-2 infection in vitro. Vero E6 cells were infected with SARS-CoV-2 at MOIs of 0.1 and 0.01 and treated with different concentrations of Qx or with PBS (controls) for 1 h, then the inoculum was changed by fresh medium and the effect was evaluated 48 h later. A representative bright field images of Vero E6 cells in each group are shown, Qx treatments protected Vero E6 cells from viral cytotoxicity. 10× magnification.
    Figure Legend Snippet: Effect of Qx against SARS-CoV-2 infection in vitro. Vero E6 cells were infected with SARS-CoV-2 at MOIs of 0.1 and 0.01 and treated with different concentrations of Qx or with PBS (controls) for 1 h, then the inoculum was changed by fresh medium and the effect was evaluated 48 h later. A representative bright field images of Vero E6 cells in each group are shown, Qx treatments protected Vero E6 cells from viral cytotoxicity. 10× magnification.

    Techniques Used: Infection, In Vitro

    Representative immunofluorescence pictures on the effect Qx treatment (CC 6.25 , CC 12.5 and CC 25 ) in the infection of SARS-CoV-2 (MOI 0.1 and 0.01) in Vero E6 cells. Infected cells with MOI 0.1 and 0.01 are shown by the columns 1 and 2 respectively, as well as with the three different CC (6.25, 12.5, 25) for Qx treatment. Non-infected control cells are in the third column. In green: SARS-CoV-2 Spike Protein S2, and nucleus was counter stained with DAPI. Scale bar, 20 μm.
    Figure Legend Snippet: Representative immunofluorescence pictures on the effect Qx treatment (CC 6.25 , CC 12.5 and CC 25 ) in the infection of SARS-CoV-2 (MOI 0.1 and 0.01) in Vero E6 cells. Infected cells with MOI 0.1 and 0.01 are shown by the columns 1 and 2 respectively, as well as with the three different CC (6.25, 12.5, 25) for Qx treatment. Non-infected control cells are in the third column. In green: SARS-CoV-2 Spike Protein S2, and nucleus was counter stained with DAPI. Scale bar, 20 μm.

    Techniques Used: Immunofluorescence, Infection, Staining

    Visualization of Qx effects on SARS-CoV-2 infection by electron microscopy. Ultrathin-section electron micrographs of Vero E6 cells infected with a SARS-CoV-2 (MOI 0.01) treated with Qx (CC 12.5 ), after 24 h pi. Non-infected Vero cells shows a normal cytoplasm with mitochondria, rough endoplasmic reticulum, and few vesicles. SARS-CoV-2 infected-Vero cells showed multiple assembled virions into double membrane endosomes or vacuoles, and in the vesicles (arrows). Infected cells treated with Qx shows multiple empty vesicles containing electron dense deposits, without viral particles as compared to cells without treatment (bar = 500 nm).
    Figure Legend Snippet: Visualization of Qx effects on SARS-CoV-2 infection by electron microscopy. Ultrathin-section electron micrographs of Vero E6 cells infected with a SARS-CoV-2 (MOI 0.01) treated with Qx (CC 12.5 ), after 24 h pi. Non-infected Vero cells shows a normal cytoplasm with mitochondria, rough endoplasmic reticulum, and few vesicles. SARS-CoV-2 infected-Vero cells showed multiple assembled virions into double membrane endosomes or vacuoles, and in the vesicles (arrows). Infected cells treated with Qx shows multiple empty vesicles containing electron dense deposits, without viral particles as compared to cells without treatment (bar = 500 nm).

    Techniques Used: Infection, Electron Microscopy

    22) Product Images from "A Polarized Cell Model for Chikungunya Virus Infection: Entry and Egress of Virus Occurs at the Apical Domain of Polarized Cells"

    Article Title: A Polarized Cell Model for Chikungunya Virus Infection: Entry and Egress of Virus Occurs at the Apical Domain of Polarized Cells

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0002661

    Polarized entry of CHIKV at apical plasma membrane domain. ( A ) The total virus yield at 24 h.p.i. of non-polarized Vero, polarized Vero C1008 and polarized HBMEC cells at an MOI of 10 was quantified by viral plaque assay. Entry of CHIKV is bi-directional in non-polarized Vero cells but occurs preferentially at the apical domain of polarized Vero C1008 and HBMEC cells. Two-tailed Student's t -test: * p
    Figure Legend Snippet: Polarized entry of CHIKV at apical plasma membrane domain. ( A ) The total virus yield at 24 h.p.i. of non-polarized Vero, polarized Vero C1008 and polarized HBMEC cells at an MOI of 10 was quantified by viral plaque assay. Entry of CHIKV is bi-directional in non-polarized Vero cells but occurs preferentially at the apical domain of polarized Vero C1008 and HBMEC cells. Two-tailed Student's t -test: * p

    Techniques Used: Viral Plaque Assay, Two Tailed Test

    Polarized release of CHIKV at apical plasma membrane domain. ( A ) Infectious virus titer of supernatants collected from the apical and basolateral chambers at 24 h.p.i. of non-polarized Vero, polarized Vero C1008 and polarized HBMEC cells at an MOI of 10 were quantified by viral plaque assays. Release of CHIKV is bi-directional in non-polarized Vero cells but occurs preferentially at the apical domain of polarized Vero C1008 and HBMEC cells. Two-tailed Student's t -test: * p
    Figure Legend Snippet: Polarized release of CHIKV at apical plasma membrane domain. ( A ) Infectious virus titer of supernatants collected from the apical and basolateral chambers at 24 h.p.i. of non-polarized Vero, polarized Vero C1008 and polarized HBMEC cells at an MOI of 10 were quantified by viral plaque assays. Release of CHIKV is bi-directional in non-polarized Vero cells but occurs preferentially at the apical domain of polarized Vero C1008 and HBMEC cells. Two-tailed Student's t -test: * p

    Techniques Used: Two Tailed Test

    Release of CHIKV upon drug treatment to elucidate viral factors involved in apical sorting of CHIKV. ( A ) The infectious virus titers were similar in the apical and basolateral chambers upon treatment with tunicamycin. Vertical bars represent one standard deviation from the mean of three readings. ( B ) Apically-infected Vero C1008 cells were treated with tunicamycin and co-labeled with antibodies against CHIKV E2 glycoprotein (green, arrows) and ZO-1 tight junction proteins apical markers (red, arrowheads). Cell nuclei were stained with DAPI (blue). Z-stacked images show the bidirectional release of CHIKV upon treatment with tunicamycin. (C) The copies number of CHIKV RNA was higher in the apical chamber than in the basolateral chamber upon apical infection with CHIKV. Upon tunicamycin treatment, the copies number of CHIKV RNA was similar in the apical and basolateral chambers.
    Figure Legend Snippet: Release of CHIKV upon drug treatment to elucidate viral factors involved in apical sorting of CHIKV. ( A ) The infectious virus titers were similar in the apical and basolateral chambers upon treatment with tunicamycin. Vertical bars represent one standard deviation from the mean of three readings. ( B ) Apically-infected Vero C1008 cells were treated with tunicamycin and co-labeled with antibodies against CHIKV E2 glycoprotein (green, arrows) and ZO-1 tight junction proteins apical markers (red, arrowheads). Cell nuclei were stained with DAPI (blue). Z-stacked images show the bidirectional release of CHIKV upon treatment with tunicamycin. (C) The copies number of CHIKV RNA was higher in the apical chamber than in the basolateral chamber upon apical infection with CHIKV. Upon tunicamycin treatment, the copies number of CHIKV RNA was similar in the apical and basolateral chambers.

    Techniques Used: Standard Deviation, Infection, Labeling, Staining

    Cell monolayer integrity post CHIKV infection at an MOI of 10. ( A ) TEER measurements of Vero, Vero C1008 and HBMEC cell monolayers were taken at 24 h.p.i.. The TEER measurements post-apical and basolateral infection were comparable to that of mock-infected cells. Vertical bars represent one standard deviation from the mean of three readings. ( B ) Immunofluorescence assays demonstrated the expression of ZO-1 tight junction proteins (green) in apically-infected and basolaterally-infected Vero, Vero C1008 and HBMEC cells at 24 h.p.i.. The expression of ZO-1 proteins in infected cells is comparable to that of mock-infected cells. (C) The FITC-dextran permeability assays demonstrated that the integrity of Vero and Vero C1008 cell monolayers remained intact at 24 h.p.i., where the permeability of the cell monolayers to FITC-dextran remained low as compared to the TNF-treated cells.
    Figure Legend Snippet: Cell monolayer integrity post CHIKV infection at an MOI of 10. ( A ) TEER measurements of Vero, Vero C1008 and HBMEC cell monolayers were taken at 24 h.p.i.. The TEER measurements post-apical and basolateral infection were comparable to that of mock-infected cells. Vertical bars represent one standard deviation from the mean of three readings. ( B ) Immunofluorescence assays demonstrated the expression of ZO-1 tight junction proteins (green) in apically-infected and basolaterally-infected Vero, Vero C1008 and HBMEC cells at 24 h.p.i.. The expression of ZO-1 proteins in infected cells is comparable to that of mock-infected cells. (C) The FITC-dextran permeability assays demonstrated that the integrity of Vero and Vero C1008 cell monolayers remained intact at 24 h.p.i., where the permeability of the cell monolayers to FITC-dextran remained low as compared to the TNF-treated cells.

    Techniques Used: Infection, Standard Deviation, Immunofluorescence, Expressing, Permeability

    Growth kinetics of CHIKV in Vero C1008 at an MOI of 10. ( A ) CHIKV-infected Vero C1008 cells were viewed under DIC microscopy to observe for any morphological changes. Extensive CPE was observed from 36 h.p.i. onwards, as shown by the spindle-shaped and round appearance of cells (black arrows). ( B ) CHIKV-infected Vero C1008 cells were fixed at various intervals post-infection and immunofluorescence assay was performed to detect for CHIKV protein expression (green). Cell nuclei were stained with DAPI (blue). High amounts of CHIKV protein expression was observed at 24 and 36 h.p.i. of Vero C1008. ( C ) Quantification of infectious virus titer by plaque assay showed an increasing trend, with a peak in infectious virus titer at 42 h.p.i.. Vertical bars represent one standard deviation from the mean of three readings.
    Figure Legend Snippet: Growth kinetics of CHIKV in Vero C1008 at an MOI of 10. ( A ) CHIKV-infected Vero C1008 cells were viewed under DIC microscopy to observe for any morphological changes. Extensive CPE was observed from 36 h.p.i. onwards, as shown by the spindle-shaped and round appearance of cells (black arrows). ( B ) CHIKV-infected Vero C1008 cells were fixed at various intervals post-infection and immunofluorescence assay was performed to detect for CHIKV protein expression (green). Cell nuclei were stained with DAPI (blue). High amounts of CHIKV protein expression was observed at 24 and 36 h.p.i. of Vero C1008. ( C ) Quantification of infectious virus titer by plaque assay showed an increasing trend, with a peak in infectious virus titer at 42 h.p.i.. Vertical bars represent one standard deviation from the mean of three readings.

    Techniques Used: Infection, Microscopy, Immunofluorescence, Expressing, Staining, Plaque Assay, Standard Deviation

    23) Product Images from "Fusion of Mature HIV-1 Particles Leads to Complete Release of a Gag-GFP-Based Content Marker and Raises the Intraviral pH"

    Article Title: Fusion of Mature HIV-1 Particles Leads to Complete Release of a Gag-GFP-Based Content Marker and Raises the Intraviral pH

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0071002

    A gain-of-signal assay for detecting the synchronized fusion of NH 4 Cl-arrested ASLV-A pseudoviruses. ASLV-A pseudoviruses carrying HIV-1 Gag-iCherry and YFP-Vpr were allowed to enter CV-1 cells stably expressing TVA950 in the presence of NH 4 Cl. Virus fusion was then initiated by replacing NH 4 Cl with HBSS, thereby quickly acidifying the endosomal and intraviral pH. (A) Depiction of the NH 4 Cl arrest-release protocol for synchronized ASLV-A fusion. NH 4 Cl blocks the ASLV-A fusion and traps YFP-Vpr/Gag-iCherry labeled pseudoviruses in endosomes by raising the endosomal pH (left). Removal of NH 4 Cl results in acidification of endosomal lumen and of viral interior, as evidenced by quenching of the YFP-Vpr signal (middle). Subsequent acid-mediated fusion with an endosome results in iCherry release and YFP-Vpr dequenching caused by re-neutralization of the intraviral pH through a fusion pore that connect the virus interior to the cytoplasm (right). (B) Snapshots of ASLV-A pseudovirus fusion triggered by removal of NH 4 Cl (see also movie S3 ). Particles co-labeled with Gag-iCherry (red) and YFP-Vpr (green) were pre-bound to cells expressing TVA950 in the cold and incubated for at 37°C for 40 min in HBSS supplemented with 70 mM NH 4 Cl. The first micrograph is taken prior to removal of NH 4 Cl (t = 0 s). The second micrograph (t = 100 s) is taken shortly after substituting NH 4 Cl with HBSS, which results in YFP quenching due to acidification of the virus interior. The third micrograph shows pseudoviruses exhibiting the loss of iCherry and concomitant appearance of bright YFP signal caused by virus fusion (marked with white circles), while the endosomal and intraviral pH still remain acidic. The fourth micrograph shows the fluorescence pattern after returning to NH 4 Cl, which re-neutralizes the endosomal/intraviral pH and causes dequenching of the YFP signal from particles that failed to undergo fusion. The particle marked by an arrow exhibited a delayed release of iCherry relative to YFP dequenching, apparently due to slow dilation of a nascent fusion pore. Scale bar 14 µm. (C) Images of a single ASLV-A pseudovirus (dashed circle) fusing after removal of NH 4 Cl. The initial drop in the YFP signal is caused by acidification of the virus interior, whereas the loss of iCherry during the HBSS perfusion (while the intraviral pH is still acidic) corresponds to virus-endosome fusion (see movie S4 ). A non-fusing particle (arrow) exhibits reversible changes in YFP but not iCherry fluorescence in response to HBSS/NH 4 Cl perfusion. Scale bar is 8 µm. (D-F) The fluorescence intensity profiles for the fusing (D) and non-fusing (F) particles from panel C. The fusion event occurring during HBSS perfusion (block arrow) is manifested in the iCherry release (red) and concomitant dequenching of YFP fluorescence (green). Changes in the intraviral and the cytosolic pH during the removal/addition of NH 4 Cl determined in separate experiments are shown by blue and pink lines, respectively (E). The double-arrow in panel E shows the predicted pH difference between the intraviral and cytosolic compartments at the time of fusion shown in panels C and D.
    Figure Legend Snippet: A gain-of-signal assay for detecting the synchronized fusion of NH 4 Cl-arrested ASLV-A pseudoviruses. ASLV-A pseudoviruses carrying HIV-1 Gag-iCherry and YFP-Vpr were allowed to enter CV-1 cells stably expressing TVA950 in the presence of NH 4 Cl. Virus fusion was then initiated by replacing NH 4 Cl with HBSS, thereby quickly acidifying the endosomal and intraviral pH. (A) Depiction of the NH 4 Cl arrest-release protocol for synchronized ASLV-A fusion. NH 4 Cl blocks the ASLV-A fusion and traps YFP-Vpr/Gag-iCherry labeled pseudoviruses in endosomes by raising the endosomal pH (left). Removal of NH 4 Cl results in acidification of endosomal lumen and of viral interior, as evidenced by quenching of the YFP-Vpr signal (middle). Subsequent acid-mediated fusion with an endosome results in iCherry release and YFP-Vpr dequenching caused by re-neutralization of the intraviral pH through a fusion pore that connect the virus interior to the cytoplasm (right). (B) Snapshots of ASLV-A pseudovirus fusion triggered by removal of NH 4 Cl (see also movie S3 ). Particles co-labeled with Gag-iCherry (red) and YFP-Vpr (green) were pre-bound to cells expressing TVA950 in the cold and incubated for at 37°C for 40 min in HBSS supplemented with 70 mM NH 4 Cl. The first micrograph is taken prior to removal of NH 4 Cl (t = 0 s). The second micrograph (t = 100 s) is taken shortly after substituting NH 4 Cl with HBSS, which results in YFP quenching due to acidification of the virus interior. The third micrograph shows pseudoviruses exhibiting the loss of iCherry and concomitant appearance of bright YFP signal caused by virus fusion (marked with white circles), while the endosomal and intraviral pH still remain acidic. The fourth micrograph shows the fluorescence pattern after returning to NH 4 Cl, which re-neutralizes the endosomal/intraviral pH and causes dequenching of the YFP signal from particles that failed to undergo fusion. The particle marked by an arrow exhibited a delayed release of iCherry relative to YFP dequenching, apparently due to slow dilation of a nascent fusion pore. Scale bar 14 µm. (C) Images of a single ASLV-A pseudovirus (dashed circle) fusing after removal of NH 4 Cl. The initial drop in the YFP signal is caused by acidification of the virus interior, whereas the loss of iCherry during the HBSS perfusion (while the intraviral pH is still acidic) corresponds to virus-endosome fusion (see movie S4 ). A non-fusing particle (arrow) exhibits reversible changes in YFP but not iCherry fluorescence in response to HBSS/NH 4 Cl perfusion. Scale bar is 8 µm. (D-F) The fluorescence intensity profiles for the fusing (D) and non-fusing (F) particles from panel C. The fusion event occurring during HBSS perfusion (block arrow) is manifested in the iCherry release (red) and concomitant dequenching of YFP fluorescence (green). Changes in the intraviral and the cytosolic pH during the removal/addition of NH 4 Cl determined in separate experiments are shown by blue and pink lines, respectively (E). The double-arrow in panel E shows the predicted pH difference between the intraviral and cytosolic compartments at the time of fusion shown in panels C and D.

    Techniques Used: Stable Transfection, Expressing, Labeling, Neutralization, Incubation, Fluorescence, Blocking Assay

    Detection of HIV-1 fusion by content release and intraviral pH-sensing assays. HIV-1 pseudoviruses bearing HXB2 Env and co-labeled with YFP-Vpr and Gag-iCherry were spinoculated onto CV-1/CD4/CXCR4 cells in the cold. Cells were washed and imaged at 37°C. (A) Images of single virus fusion leading to loss of the iCherry signal (red). Time stamps (in min:sec) indicate the time after shifting the cells to 37°C (see also movie S1 ). Scale bar is 2 µm. Cartoons above and below the image panel illustrate that this labeling approach does not discern between virus-endosomes (top) and virus-plasma membrane (bottom) fusion. (B) Mean fluorescence intensities of YFP and iCherry signals for the particle shown in panel A. Virus-cell fusion results in simultaneous YFP fluorescence dequenching and loss of the iCherry signal (arrow). (C) An example of a particle that fails to fuse. The slow decrease in the YFP fluorescence is caused by acidification of the virus’ interior, which most likely reflects the pH drop in endosomal compartments. (D) The kinetics of HIV-1 fusion with cells measured as the distribution of waiting times from raising the temperature to loss of iCherry (dark red circles). For comparison, the kinetic of ASLV-A Env-mediated fusion of HIV-1-based particles with CV1 cells expressing TVA950 is also shown (open circles).
    Figure Legend Snippet: Detection of HIV-1 fusion by content release and intraviral pH-sensing assays. HIV-1 pseudoviruses bearing HXB2 Env and co-labeled with YFP-Vpr and Gag-iCherry were spinoculated onto CV-1/CD4/CXCR4 cells in the cold. Cells were washed and imaged at 37°C. (A) Images of single virus fusion leading to loss of the iCherry signal (red). Time stamps (in min:sec) indicate the time after shifting the cells to 37°C (see also movie S1 ). Scale bar is 2 µm. Cartoons above and below the image panel illustrate that this labeling approach does not discern between virus-endosomes (top) and virus-plasma membrane (bottom) fusion. (B) Mean fluorescence intensities of YFP and iCherry signals for the particle shown in panel A. Virus-cell fusion results in simultaneous YFP fluorescence dequenching and loss of the iCherry signal (arrow). (C) An example of a particle that fails to fuse. The slow decrease in the YFP fluorescence is caused by acidification of the virus’ interior, which most likely reflects the pH drop in endosomal compartments. (D) The kinetics of HIV-1 fusion with cells measured as the distribution of waiting times from raising the temperature to loss of iCherry (dark red circles). For comparison, the kinetic of ASLV-A Env-mediated fusion of HIV-1-based particles with CV1 cells expressing TVA950 is also shown (open circles).

    Techniques Used: Labeling, Size-exclusion Chromatography, Fluorescence, Expressing

    24) Product Images from "Synchronized Retrovirus Fusion in Cells Expressing Alternative Receptor Isoforms Releases the Viral Core into Distinct Sub-cellular Compartments"

    Article Title: Synchronized Retrovirus Fusion in Cells Expressing Alternative Receptor Isoforms Releases the Viral Core into Distinct Sub-cellular Compartments

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1002694

    Arrest and synchronous triggering of ASLV pseudovirus fusion with endosomes using NH 4 Cl. (A) Micrograph showing single ASLV pseudoviruses co-labeled with Gag-GFP (green) and DiD (red) following the incubation with cells expressing TVA800 for 40 min at 37°C in isotonic HBSS supplemented with 70 mM NH 4 Cl (left panel). Removal of NH 4 Cl through perfusion with HBSS caused a marked decrease in the GFP signal, but not in DiD fluorescence (middle). Upon returning to NH 4 Cl (right), the GFP fluorescence of fusion-incompetent particles fully recovered (#1, arrowhead), whereas the signal from fused particles remained undetectable (#2, arrow). The viral core release into the cytosol is manifested in spatial separation of green and red puncta (#3, double arrowhead). Cell nuclei are labeled with Hoescht (blue). Scale bar is 15 µm. (B–G) ASLV pseudoviruses co-labeled with Gag-GFP and DiD were internalized by CV-1 cells expressing TVA950 (B, D, F) or TVA800 (C, E, G) in the presence of NH 4 Cl, and virus-endosome fusion was initiated by perfusion with HBSS, as described in Materials and Methods . (B, C) The fluorescence intensity profiles corresponding to particles that failed to fuse following the arrest/release protocol, as evidenced by complete recovery of the GFP signal. (D, E) Examples of complete loss of the GFP marker from virions. (F, G) Partial release of the content marker. Cells were initially perfused with 70 mM NH 4 Cl in HBSS (white thick horizontal bars at the top of each graph) followed by perfusion with plain HBSS for 2 min (black horizontal bars) and returned to NH 4 Cl. The mean intensities of GFP and DiD fluorescence from single particles are shown (green and red circles, respectively). Pink lines show the changes in the intraviral pH upon removal of NH 4 Cl (for details, see Figures. S1 and S2 ). Kymographs illustrating the time-dependent changes in the mean GFP and DID signals (overlaid) are also shown above each plot. An irreversible transition from yellow (co-localized GFP and DiD signals) to red (DiD only) manifests the release of the GFP-based content marker into the cytosol. Diagrams of the fusion outcomes (reversible GFP quenching by acidic pH, full and partial release of the content marker) for respective panels are shown on the right.
    Figure Legend Snippet: Arrest and synchronous triggering of ASLV pseudovirus fusion with endosomes using NH 4 Cl. (A) Micrograph showing single ASLV pseudoviruses co-labeled with Gag-GFP (green) and DiD (red) following the incubation with cells expressing TVA800 for 40 min at 37°C in isotonic HBSS supplemented with 70 mM NH 4 Cl (left panel). Removal of NH 4 Cl through perfusion with HBSS caused a marked decrease in the GFP signal, but not in DiD fluorescence (middle). Upon returning to NH 4 Cl (right), the GFP fluorescence of fusion-incompetent particles fully recovered (#1, arrowhead), whereas the signal from fused particles remained undetectable (#2, arrow). The viral core release into the cytosol is manifested in spatial separation of green and red puncta (#3, double arrowhead). Cell nuclei are labeled with Hoescht (blue). Scale bar is 15 µm. (B–G) ASLV pseudoviruses co-labeled with Gag-GFP and DiD were internalized by CV-1 cells expressing TVA950 (B, D, F) or TVA800 (C, E, G) in the presence of NH 4 Cl, and virus-endosome fusion was initiated by perfusion with HBSS, as described in Materials and Methods . (B, C) The fluorescence intensity profiles corresponding to particles that failed to fuse following the arrest/release protocol, as evidenced by complete recovery of the GFP signal. (D, E) Examples of complete loss of the GFP marker from virions. (F, G) Partial release of the content marker. Cells were initially perfused with 70 mM NH 4 Cl in HBSS (white thick horizontal bars at the top of each graph) followed by perfusion with plain HBSS for 2 min (black horizontal bars) and returned to NH 4 Cl. The mean intensities of GFP and DiD fluorescence from single particles are shown (green and red circles, respectively). Pink lines show the changes in the intraviral pH upon removal of NH 4 Cl (for details, see Figures. S1 and S2 ). Kymographs illustrating the time-dependent changes in the mean GFP and DID signals (overlaid) are also shown above each plot. An irreversible transition from yellow (co-localized GFP and DiD signals) to red (DiD only) manifests the release of the GFP-based content marker into the cytosol. Diagrams of the fusion outcomes (reversible GFP quenching by acidic pH, full and partial release of the content marker) for respective panels are shown on the right.

    Techniques Used: Labeling, Incubation, Expressing, Fluorescence, Marker

    25) Product Images from "A subpopulation of arenavirus nucleoprotein localizes to mitochondria"

    Article Title: A subpopulation of arenavirus nucleoprotein localizes to mitochondria

    Journal: Scientific Reports

    doi: 10.1038/s41598-021-99887-5

    In vitro mitochondria import assay of arenaviral NPs. ( a – c ) The in vitro import into mitochondria was determined for a known chimeric mitochondrial protein, Su9-DHFR, used as positive control for the assay. The fusion protein was synthesized using [ 35 S]-methionine in a rabbit reticulocyte lysate, from its sequence cloned in a pGEM4Z vector, flanking the SP6 RNA promoter. The hybrid protein was imported into freshly isolated mitochondria of monkey Vero E6 cells, at 37 °C ( a ), Boa constrictor kidney (I/1Ki) cells, at both 37 °C and 30 °C ( b ), and Python regius heart (VI/1Hz) cells, at 30 °C ( c ), as indicated by the presence at different time points of three distinct translocation forms: precursor, intermediate and mature forms (black arrows). ( d – h ) The in vitro translocation into freshly isolated Boa constrictor I/1Ki mitochondria was assessed for HISV-1 NP, at 30 °C ( d ), UHV-1 NP, at 30 °C ( e ) and 37 °C ( f ), wt and mutUGV-1 NPs, at 30 °C ( g ) and HA-tagged JUNV and LCMV NPs, at 37 °C ( h ). Radiolabelled NPs were in vitro synthesized using [ 35 S]-methionine in a rabbit reticulocyte lysate, from their ORFs cloned into pGEM4Z ( d–g ) or pCR4Blunt-TOPO ( h ) vectors, flanking the SP6 ( d–g ) or T7 ( h ) promoter. Protein signals were determined through autoradiographic detection. CCCP: mitochondrial protein import blocker by inducing mitochondrial membrane potential dissipation. proK: leading to degradation of non-imported proteins. Full-length autoradiographies are presented in Supplementary Fig. S12 .
    Figure Legend Snippet: In vitro mitochondria import assay of arenaviral NPs. ( a – c ) The in vitro import into mitochondria was determined for a known chimeric mitochondrial protein, Su9-DHFR, used as positive control for the assay. The fusion protein was synthesized using [ 35 S]-methionine in a rabbit reticulocyte lysate, from its sequence cloned in a pGEM4Z vector, flanking the SP6 RNA promoter. The hybrid protein was imported into freshly isolated mitochondria of monkey Vero E6 cells, at 37 °C ( a ), Boa constrictor kidney (I/1Ki) cells, at both 37 °C and 30 °C ( b ), and Python regius heart (VI/1Hz) cells, at 30 °C ( c ), as indicated by the presence at different time points of three distinct translocation forms: precursor, intermediate and mature forms (black arrows). ( d – h ) The in vitro translocation into freshly isolated Boa constrictor I/1Ki mitochondria was assessed for HISV-1 NP, at 30 °C ( d ), UHV-1 NP, at 30 °C ( e ) and 37 °C ( f ), wt and mutUGV-1 NPs, at 30 °C ( g ) and HA-tagged JUNV and LCMV NPs, at 37 °C ( h ). Radiolabelled NPs were in vitro synthesized using [ 35 S]-methionine in a rabbit reticulocyte lysate, from their ORFs cloned into pGEM4Z ( d–g ) or pCR4Blunt-TOPO ( h ) vectors, flanking the SP6 ( d–g ) or T7 ( h ) promoter. Protein signals were determined through autoradiographic detection. CCCP: mitochondrial protein import blocker by inducing mitochondrial membrane potential dissipation. proK: leading to degradation of non-imported proteins. Full-length autoradiographies are presented in Supplementary Fig. S12 .

    Techniques Used: In Vitro, Positive Control, Synthesized, Sequencing, Clone Assay, Plasmid Preparation, Isolation, Translocation Assay

    Immunoblotting studies on arenaviral NPs in transfected mammalian cells. ( a – c ) Immunoblotting analyses of whole-cell lysates obtained from monkey Vero E6 cells transfected with a construct expressing either wt or mutUGV1-NP-FLAG, or UHV1-NP-FLAG ( a ), wt or mutJUNV-NP-HA, or LCMV-NP-HA ( b ), HISV1-NP-FLAG ( c ), at three dpt after incubation at either 37 °C or 30 °C. Non-transfected (Mock) samples are provided as negative controls. 40 µg of protein per sample were loaded on standard SDS-PAGE gels, followed by immunoblotting. The nitrocellulose membranes were incubated sequentially with the following antibodies in the presented order: (1) mouse anti-FLAG tag 1:500 ( a , c ) or mouse anti-HA tag 1:500 ( b ); (2) rabbit anti-MTS-NP 1:200 ( a , b ) or rabbit anti-Hartmani-NP 1:500 ( c ); (3) mouse anti-tubulin 1:500 ( a–c ). Anti-tubulin specific signal at known molecular weight did not require membrane stripping. Tubulin (in red, secondary antibody: IRDye 680RD Donkey anti-mouse) was used as a reference for loading. Reptarenavirus and hartmanivirus (65–68 kDa), and mammarenavirus (63–65 kDa) NPs are indicated (black arrows). ( a ) Left panel: FLAG tag in red (secondary antibody: IRDye 680RD Donkey anti-mouse); middle panel: MTS-NP in green (secondary antibody: IRDye 800CW Donkey anti-rabbit); right panel: merged image. ( b ) Left panel: HA tag in red (secondary antibody: IRDye 680RD Donkey anti-mouse); middle panel: MTS-NP in green (secondary antibody: IRDye 800CW Donkey anti-rabbit); right panel: merged image. ( c ) Left panel: FLAG tag in red (secondary antibody: IRDye 680RD Donkey anti-mouse); middle panel: Hartmani-NP in green (secondary antibody: IRDye 800CW Donkey anti-rabbit); right panel: merged image. Immunodetection was performed using the Odyssey Infrared Imaging System (LICOR, Biosciences) providing also the molecular marker (Precision Plus Protein Dual Color Standards, Bio-Rad) used. Full-length blots are presented in Supplementary Fig. S8 .
    Figure Legend Snippet: Immunoblotting studies on arenaviral NPs in transfected mammalian cells. ( a – c ) Immunoblotting analyses of whole-cell lysates obtained from monkey Vero E6 cells transfected with a construct expressing either wt or mutUGV1-NP-FLAG, or UHV1-NP-FLAG ( a ), wt or mutJUNV-NP-HA, or LCMV-NP-HA ( b ), HISV1-NP-FLAG ( c ), at three dpt after incubation at either 37 °C or 30 °C. Non-transfected (Mock) samples are provided as negative controls. 40 µg of protein per sample were loaded on standard SDS-PAGE gels, followed by immunoblotting. The nitrocellulose membranes were incubated sequentially with the following antibodies in the presented order: (1) mouse anti-FLAG tag 1:500 ( a , c ) or mouse anti-HA tag 1:500 ( b ); (2) rabbit anti-MTS-NP 1:200 ( a , b ) or rabbit anti-Hartmani-NP 1:500 ( c ); (3) mouse anti-tubulin 1:500 ( a–c ). Anti-tubulin specific signal at known molecular weight did not require membrane stripping. Tubulin (in red, secondary antibody: IRDye 680RD Donkey anti-mouse) was used as a reference for loading. Reptarenavirus and hartmanivirus (65–68 kDa), and mammarenavirus (63–65 kDa) NPs are indicated (black arrows). ( a ) Left panel: FLAG tag in red (secondary antibody: IRDye 680RD Donkey anti-mouse); middle panel: MTS-NP in green (secondary antibody: IRDye 800CW Donkey anti-rabbit); right panel: merged image. ( b ) Left panel: HA tag in red (secondary antibody: IRDye 680RD Donkey anti-mouse); middle panel: MTS-NP in green (secondary antibody: IRDye 800CW Donkey anti-rabbit); right panel: merged image. ( c ) Left panel: FLAG tag in red (secondary antibody: IRDye 680RD Donkey anti-mouse); middle panel: Hartmani-NP in green (secondary antibody: IRDye 800CW Donkey anti-rabbit); right panel: merged image. Immunodetection was performed using the Odyssey Infrared Imaging System (LICOR, Biosciences) providing also the molecular marker (Precision Plus Protein Dual Color Standards, Bio-Rad) used. Full-length blots are presented in Supplementary Fig. S8 .

    Techniques Used: Transfection, Construct, Expressing, Incubation, SDS Page, FLAG-tag, Molecular Weight, Stripping Membranes, Immunodetection, Imaging, Marker

    IF studies on arenaviral NPs in transfected boid and mammalian cells. Double IF images of Boa constrictor V/4Br cells incubated at 30 °C ( a , b ) or of monkey Vero E6 cells incubated at 37 °C ( c , d ), transfected with a construct expressing either wt or mutUGV1-NP-FLAG, UHV1-NP-FLAG, or HISV1-NP-FLAG ( a , c ), and wt or mutJUNV-NP-HA, or LCMV-NP-HA ( b , d ) at three dpt. Non-transfected (Mock) cells served as controls. ( a , c ) The panels from left: FLAG tag in red (secondary antibody: AlexaFluor 594 goat anti-rabbit), mitochondrial marker in green (secondary antibody: AlexaFluor 488 goat anti-mouse), nuclei in blue (DAPI), and a merged image. ( b , d ) The panels from left: HA tag in red (secondary antibody: AlexaFluor 594 goat anti-rabbit), mitochondrial marker in green (secondary antibody: AlexaFluor 488 goat anti-mouse), nuclei in blue (DAPI), and a merged image.
    Figure Legend Snippet: IF studies on arenaviral NPs in transfected boid and mammalian cells. Double IF images of Boa constrictor V/4Br cells incubated at 30 °C ( a , b ) or of monkey Vero E6 cells incubated at 37 °C ( c , d ), transfected with a construct expressing either wt or mutUGV1-NP-FLAG, UHV1-NP-FLAG, or HISV1-NP-FLAG ( a , c ), and wt or mutJUNV-NP-HA, or LCMV-NP-HA ( b , d ) at three dpt. Non-transfected (Mock) cells served as controls. ( a , c ) The panels from left: FLAG tag in red (secondary antibody: AlexaFluor 594 goat anti-rabbit), mitochondrial marker in green (secondary antibody: AlexaFluor 488 goat anti-mouse), nuclei in blue (DAPI), and a merged image. ( b , d ) The panels from left: HA tag in red (secondary antibody: AlexaFluor 594 goat anti-rabbit), mitochondrial marker in green (secondary antibody: AlexaFluor 488 goat anti-mouse), nuclei in blue (DAPI), and a merged image.

    Techniques Used: Transfection, Incubation, Construct, Expressing, FLAG-tag, Marker

    26) Product Images from "Development of a live attenuated trivalent porcine rotavirus A vaccine against disease caused by recent strains most prevalent in South Korea"

    Article Title: Development of a live attenuated trivalent porcine rotavirus A vaccine against disease caused by recent strains most prevalent in South Korea

    Journal: Veterinary Research

    doi: 10.1186/s13567-018-0619-6

    Electropherogram (PAGE) of selected passages in MA104 cells of three porcine RVA strains. The RNA genomic segments of three vaccine strains from different passage numbers were separated by PAGE and visualized using silver staining. The original virulent strains and each passage of 174-1 ( A ), K71 ( B ), and PRG942 ( C ) demonstrated typical RVA’s RNA segment patterns of 4-2-3-2 and maintained their own patterns throughout the serial passages. Lane 1, original virulent strain; lane 2, 10 th passage; lane 3, 20 th passage; lane 4, 40 th passage; lane 5, 60 th passage; lane 6, 80 th passage.
    Figure Legend Snippet: Electropherogram (PAGE) of selected passages in MA104 cells of three porcine RVA strains. The RNA genomic segments of three vaccine strains from different passage numbers were separated by PAGE and visualized using silver staining. The original virulent strains and each passage of 174-1 ( A ), K71 ( B ), and PRG942 ( C ) demonstrated typical RVA’s RNA segment patterns of 4-2-3-2 and maintained their own patterns throughout the serial passages. Lane 1, original virulent strain; lane 2, 10 th passage; lane 3, 20 th passage; lane 4, 40 th passage; lane 5, 60 th passage; lane 6, 80 th passage.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Silver Staining

    27) Product Images from "Sendai Virus Infection Induces Apoptosis through Activation of Caspase-8 (FLICE) and Caspase-3 (CPP32)"

    Article Title: Sendai Virus Infection Induces Apoptosis through Activation of Caspase-8 (FLICE) and Caspase-3 (CPP32)

    Journal: Journal of Virology

    doi:

    DNA fragmentation induced by SV infection of CV-1 cells. Lanes 2 to 4, DNA preparations from productively infected cells at different time points p.i.; lane 1, DNA marker; lane 5, preparation from uninfected control cells.
    Figure Legend Snippet: DNA fragmentation induced by SV infection of CV-1 cells. Lanes 2 to 4, DNA preparations from productively infected cells at different time points p.i.; lane 1, DNA marker; lane 5, preparation from uninfected control cells.

    Techniques Used: Infection, Marker

    In situ detection of apoptosis in infected CV-1 cells. Apoptotic DNA degradation was visualized by TUNEL staining and subsequent alkaline phosphatase staining as described in Materials and Methods. (A) Intense dark staining of CV-1 cells 24 h p.i.; (B) uninfected CV-1 cells.
    Figure Legend Snippet: In situ detection of apoptosis in infected CV-1 cells. Apoptotic DNA degradation was visualized by TUNEL staining and subsequent alkaline phosphatase staining as described in Materials and Methods. (A) Intense dark staining of CV-1 cells 24 h p.i.; (B) uninfected CV-1 cells.

    Techniques Used: In Situ, Infection, TUNEL Assay, Staining

    Detection of hypodiploid DNA in SV-infected cells and inhibition by caspase inhibitor z-VAD. CV-1 cells were infected by SV (MOI, 10) and incubated with 0 (A, B, and C) or 100 (D, E, and F) μM of the caspase inhibitor z-VAD-fmk. Shown are the results of cytometric analysis at 36 and 60 h p.i. as well as the analysis of uninfected CV-1 cells (control) and CV-1 cells incubated with z-VAD-fmk for 60 h [control (+ z-VAD)] to exclude the toxic effects of z-VAD-fmk on these cells. The proportion of sub-2N DNA is indicated in the histograms.
    Figure Legend Snippet: Detection of hypodiploid DNA in SV-infected cells and inhibition by caspase inhibitor z-VAD. CV-1 cells were infected by SV (MOI, 10) and incubated with 0 (A, B, and C) or 100 (D, E, and F) μM of the caspase inhibitor z-VAD-fmk. Shown are the results of cytometric analysis at 36 and 60 h p.i. as well as the analysis of uninfected CV-1 cells (control) and CV-1 cells incubated with z-VAD-fmk for 60 h [control (+ z-VAD)] to exclude the toxic effects of z-VAD-fmk on these cells. The proportion of sub-2N DNA is indicated in the histograms.

    Techniques Used: Infection, Inhibition, Incubation

    ); initial cleavage generates p43 and p12 intermediates, followed by further processing to the active p18 and p10 subunits and the FLICE prodomain p26. (B and C) HepG2 (B) and CV-1 (C) cells were infected with SV (MOI, 10), and FLICE and cleavage products p43 and p18 were detected by Western blot analysis at the indicated times p.i. Lanes 1 and 2, preparations from uninfected cells (control cells) and, as a positive control, from uninfected cells incubated with CD95L, respectively.
    Figure Legend Snippet: ); initial cleavage generates p43 and p12 intermediates, followed by further processing to the active p18 and p10 subunits and the FLICE prodomain p26. (B and C) HepG2 (B) and CV-1 (C) cells were infected with SV (MOI, 10), and FLICE and cleavage products p43 and p18 were detected by Western blot analysis at the indicated times p.i. Lanes 1 and 2, preparations from uninfected cells (control cells) and, as a positive control, from uninfected cells incubated with CD95L, respectively.

    Techniques Used: Infection, Western Blot, Positive Control, Incubation

    28) Product Images from "The herpes simplex virus type I deamidase enhances propagation but is dispensable for retrograde axonal transport into the nervous system"

    Article Title: The herpes simplex virus type I deamidase enhances propagation but is dispensable for retrograde axonal transport into the nervous system

    Journal: bioRxiv

    doi: 10.1101/704338

    Mutation of pUL37 C819 delays invasion of the trigeminal ganglia upon ocular inoculation of mice. Mice were infected with HSV-1 encoding the pUL25/mCherry capsid fusion on both eyes following dual corneal scarification. (A) Tear film was sampled by swabbing each eye at the indicated times post infection. The data is a compilation of two experiments: four animals were used for the 12-20 hpi samples, and five animals were used for the 24-72 hpi samples. (B) Infectious HSV-1 (black bars) and HSV-1 DNA (grey bars) were recovered from individual TGs harvested and homogenized at 4 dpi. HSV-1 DNA levels were measured by qPCR and expressed as a fold change relative to mock-infected. Samples from each set of infections are presented in order of the titer recovered. (C) Titers were determined from TGs of additional mice. The mean titer of each virus is indicated by a red bar (10 TGs per virus from 5 mice; pairs of TGs from same animal are shaded equivalently). (D) Recovered C819S virus from the TG indicated in panel B (†) was examined for plaque diameter (left) and by sequence analysis (right). (*, p
    Figure Legend Snippet: Mutation of pUL37 C819 delays invasion of the trigeminal ganglia upon ocular inoculation of mice. Mice were infected with HSV-1 encoding the pUL25/mCherry capsid fusion on both eyes following dual corneal scarification. (A) Tear film was sampled by swabbing each eye at the indicated times post infection. The data is a compilation of two experiments: four animals were used for the 12-20 hpi samples, and five animals were used for the 24-72 hpi samples. (B) Infectious HSV-1 (black bars) and HSV-1 DNA (grey bars) were recovered from individual TGs harvested and homogenized at 4 dpi. HSV-1 DNA levels were measured by qPCR and expressed as a fold change relative to mock-infected. Samples from each set of infections are presented in order of the titer recovered. (C) Titers were determined from TGs of additional mice. The mean titer of each virus is indicated by a red bar (10 TGs per virus from 5 mice; pairs of TGs from same animal are shaded equivalently). (D) Recovered C819S virus from the TG indicated in panel B (†) was examined for plaque diameter (left) and by sequence analysis (right). (*, p

    Techniques Used: Mutagenesis, Mouse Assay, Infection, Real-time Polymerase Chain Reaction, Sequencing

    The pUL37 deamidase supports infection at low multiplicity and viral spread. (A) HSV-1 single-step propagation kinetics were determined by counting plaque-forming units harvested from Vero cells (Cells) and the corresponding supernatant (Sups) at the times indicated. (B) HSV-1 plaque sizes on NHDF cells at 32 hpi (left) and Vero cells at 72 hpi (right) were compiled across three independent experiments and plotted as a percentage of wild type. (C) Vero cells infected with HSV-1 encoding a tdTomato-NLS reporter at MOI 5 (left) or at the designated MOI (right) were harvested at the indicated times and scored for red fluorescence by flow cytometry. Error bars are s.d. (****, p
    Figure Legend Snippet: The pUL37 deamidase supports infection at low multiplicity and viral spread. (A) HSV-1 single-step propagation kinetics were determined by counting plaque-forming units harvested from Vero cells (Cells) and the corresponding supernatant (Sups) at the times indicated. (B) HSV-1 plaque sizes on NHDF cells at 32 hpi (left) and Vero cells at 72 hpi (right) were compiled across three independent experiments and plotted as a percentage of wild type. (C) Vero cells infected with HSV-1 encoding a tdTomato-NLS reporter at MOI 5 (left) or at the designated MOI (right) were harvested at the indicated times and scored for red fluorescence by flow cytometry. Error bars are s.d. (****, p

    Techniques Used: Infection, Fluorescence, Flow Cytometry

    The pUL37 deamidase is dispensable for retrograde axonal transport. Primary sensory neurons were infected with HSV-1 encoding the pUL25/mCherry fusion and the indicated pUL37 allele, and transport dynamics in axons were monitored for the first hour post infection. (A) Fraction of time capsids moved in the retrograde direction, anterograde direction, or were stopped. (B) Average number of stops and reversals exhibited by capsids. (C) Distributions of forward run velocities and forward run distances of individual capsids in axons during the first hour post infection. (D) Representative images of capsids at nuclear rims of rat DRGs at 3-4 hpi. For panels A-D, more than thirty capsids were analyzed per experiment across three biological replicates.
    Figure Legend Snippet: The pUL37 deamidase is dispensable for retrograde axonal transport. Primary sensory neurons were infected with HSV-1 encoding the pUL25/mCherry fusion and the indicated pUL37 allele, and transport dynamics in axons were monitored for the first hour post infection. (A) Fraction of time capsids moved in the retrograde direction, anterograde direction, or were stopped. (B) Average number of stops and reversals exhibited by capsids. (C) Distributions of forward run velocities and forward run distances of individual capsids in axons during the first hour post infection. (D) Representative images of capsids at nuclear rims of rat DRGs at 3-4 hpi. For panels A-D, more than thirty capsids were analyzed per experiment across three biological replicates.

    Techniques Used: Infection

    The pUL37 residues C819 and C850 are conserved within the simplexvirus genera. Alignment of the deamidase region of pUL37 across 24 members of the alpha-herpesvirinae subfamily spanning the simplexvirus and varicellovirus genera. The HSV-1 residues C819 and C850 are circled in blue.
    Figure Legend Snippet: The pUL37 residues C819 and C850 are conserved within the simplexvirus genera. Alignment of the deamidase region of pUL37 across 24 members of the alpha-herpesvirinae subfamily spanning the simplexvirus and varicellovirus genera. The HSV-1 residues C819 and C850 are circled in blue.

    Techniques Used:

    29) Product Images from "Novel antiviral activity of PAD inhibitors against human beta-coronaviruses HCoV-OC43 and SARS-CoV-2"

    Article Title: Novel antiviral activity of PAD inhibitors against human beta-coronaviruses HCoV-OC43 and SARS-CoV-2

    Journal: Antiviral Research

    doi: 10.1016/j.antiviral.2022.105278

    The pan-PAD inhibitors Cl-A and BB-Cl block β-coronavirus replication in Vero-E6 cells. (A, B) Dose-response curves of the pan-PAD inhibitors Cl-A (A) and BB-Cl (B) in VERO-E6 cells infected with HCoV-OC43 (MOI 0.1). After 72 hpi, the viral load was determined by real-time RT-PCR and values were normalized to those for DMSO-treated cells value (0 in the x axis) set to 1. Values represent the mean ± SEM of three independent experiments. (C) Rh-PG and Western blot analysis of total protein extract from mock – or HCoV-OC43-infected VERO-E6 cells (MOI 1) treated with Cl-A (300 μM) or DMSO. One representative gel of three independent experiments is shown. (D, E) Dose-response curves of Cl-A (D) or BB-Cl (E) treatment of SARS-CoV2-infected VERO-E6 cells (MOI 0.1). After 72 hpi, the viral load was determined by real-time RT-PCR and values were normalized to those for DMSO-treated cells value (0 in the x axis) set to 1. Values are expressed as mean ± SEM of three independent experiments. (F) Rh-PG and Western blot analysis of total protein extract of mock- or SARS-CoV- infected VERO-E6 cells (MOI 1) and with Cl-A (300 μM) or DMSO. One representative gel of three independent experiments is shown. (G) Viral productions from the same experiment described in F were collected at 72 hpi and analyzed by plaque assay. Values are expressed as mean ± SEM of three independent experiments. P
    Figure Legend Snippet: The pan-PAD inhibitors Cl-A and BB-Cl block β-coronavirus replication in Vero-E6 cells. (A, B) Dose-response curves of the pan-PAD inhibitors Cl-A (A) and BB-Cl (B) in VERO-E6 cells infected with HCoV-OC43 (MOI 0.1). After 72 hpi, the viral load was determined by real-time RT-PCR and values were normalized to those for DMSO-treated cells value (0 in the x axis) set to 1. Values represent the mean ± SEM of three independent experiments. (C) Rh-PG and Western blot analysis of total protein extract from mock – or HCoV-OC43-infected VERO-E6 cells (MOI 1) treated with Cl-A (300 μM) or DMSO. One representative gel of three independent experiments is shown. (D, E) Dose-response curves of Cl-A (D) or BB-Cl (E) treatment of SARS-CoV2-infected VERO-E6 cells (MOI 0.1). After 72 hpi, the viral load was determined by real-time RT-PCR and values were normalized to those for DMSO-treated cells value (0 in the x axis) set to 1. Values are expressed as mean ± SEM of three independent experiments. (F) Rh-PG and Western blot analysis of total protein extract of mock- or SARS-CoV- infected VERO-E6 cells (MOI 1) and with Cl-A (300 μM) or DMSO. One representative gel of three independent experiments is shown. (G) Viral productions from the same experiment described in F were collected at 72 hpi and analyzed by plaque assay. Values are expressed as mean ± SEM of three independent experiments. P

    Techniques Used: Blocking Assay, Infection, Quantitative RT-PCR, Western Blot, Plaque Assay

    30) Product Images from "SUMOylation Attenuates the Function of PGC-1? *"

    Article Title: SUMOylation Attenuates the Function of PGC-1? *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.038943

    SUMOylation is not linked to acetylation, phosphorylation or proteasomal degradation of PGC-1α. The effect of SIRT1 inhibitor NAM on the SUMOylation of PGC-1α is shown. A and B , HeLa ( A ) or COS-1 cells ( B ) on 6-well plates were transfected
    Figure Legend Snippet: SUMOylation is not linked to acetylation, phosphorylation or proteasomal degradation of PGC-1α. The effect of SIRT1 inhibitor NAM on the SUMOylation of PGC-1α is shown. A and B , HeLa ( A ) or COS-1 cells ( B ) on 6-well plates were transfected

    Techniques Used: Pyrolysis Gas Chromatography, Transfection

    Repression of PGC-1α activity by RIP140. A , coimmunoprecipitation of PGC-1α1-215 and RIP140. COS-1 cells were transfected with 650 ng of pCMX-Gal4-PGC-1α1-215 or its mutants, pSG5-HA-RIP140 and pSG5-His-SUMO-1. The cells were lysed
    Figure Legend Snippet: Repression of PGC-1α activity by RIP140. A , coimmunoprecipitation of PGC-1α1-215 and RIP140. COS-1 cells were transfected with 650 ng of pCMX-Gal4-PGC-1α1-215 or its mutants, pSG5-HA-RIP140 and pSG5-His-SUMO-1. The cells were lysed

    Techniques Used: Pyrolysis Gas Chromatography, Activity Assay, Transfection

    PIAS1 and -3 enhance and SENP1 and -2 reverse the SUMO modification of PGC-1α. A , PIAS3 markedly promotes SUMOylation of PGC-1α. The COS-1 cells were transfected with 1 μg of pcDNA-F-PGC-1α in the presence of 0.4 μg
    Figure Legend Snippet: PIAS1 and -3 enhance and SENP1 and -2 reverse the SUMO modification of PGC-1α. A , PIAS3 markedly promotes SUMOylation of PGC-1α. The COS-1 cells were transfected with 1 μg of pcDNA-F-PGC-1α in the presence of 0.4 μg

    Techniques Used: Modification, Pyrolysis Gas Chromatography, Transfection

    31) Product Images from "Characterization of Rotavirus RNAs That Activate Innate Immune Signaling through the RIG-I-Like Receptors"

    Article Title: Characterization of Rotavirus RNAs That Activate Innate Immune Signaling through the RIG-I-Like Receptors

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0069825

    Lack of detectable dsRNA in and isolation of immunostimulatory RNAs from RV-infected cells. (A) MA104 cells in chamber slides were mock-infected, infected with RRV at an estimated MOI of 10, or transfected with 1 μg/well of polyI:C and fixed after a 6-hour (hr) incubation. J2 monoclonal antibody was used to detect dsRNA (red) and NSP5 polyclonal antibody was used to visualize infected cells (green). DAPI was used to stain cell nuclei (blue). (B) Wild type murine embryonic fibroblasts (MEFs) were mock-transfected or transfected with 500 ng/well of in vivo 6 hr large RNAs, in vivo 1 hr large RNAs, or polyI:C. Approximately 21 hours later, mouse IFN-beta ELISA was used to measure the concentration of secreted IFN-beta protein in the cell media. Bars show the average IFN-beta concentration plus standard deviation. ND, not detected.
    Figure Legend Snippet: Lack of detectable dsRNA in and isolation of immunostimulatory RNAs from RV-infected cells. (A) MA104 cells in chamber slides were mock-infected, infected with RRV at an estimated MOI of 10, or transfected with 1 μg/well of polyI:C and fixed after a 6-hour (hr) incubation. J2 monoclonal antibody was used to detect dsRNA (red) and NSP5 polyclonal antibody was used to visualize infected cells (green). DAPI was used to stain cell nuclei (blue). (B) Wild type murine embryonic fibroblasts (MEFs) were mock-transfected or transfected with 500 ng/well of in vivo 6 hr large RNAs, in vivo 1 hr large RNAs, or polyI:C. Approximately 21 hours later, mouse IFN-beta ELISA was used to measure the concentration of secreted IFN-beta protein in the cell media. Bars show the average IFN-beta concentration plus standard deviation. ND, not detected.

    Techniques Used: Isolation, Infection, Transfection, Incubation, Staining, In Vivo, Enzyme-linked Immunosorbent Assay, Concentration Assay, Standard Deviation

    32) Product Images from "NSs amyloid formation is associated with the virulence of Rift Valley fever virus in mice"

    Article Title: NSs amyloid formation is associated with the virulence of Rift Valley fever virus in mice

    Journal: Nature Communications

    doi: 10.1038/s41467-020-17101-y

    Nuclear NSs filaments are bundles of many individual thin fibrils. a Human U-87 MG cells were exposed to Rift Valley fever virus strain ZH548 (RVFV) or its counterpart devoid of the NSs sequence (RVFV ΔNSs), both at MOI ~5. Infected cells were imaged by confocal microscopy after labeling of nuclei with Hoechst (blue) and immunofluorescence staining of the RVFV proteins NSs (green) and N (red). Images are representative of three independent experiments. Scale bar, 5 µm; pi post-infection. b Green monkey Vero cells were infected with RVFV at a MOI of 5 for 16 h. Samples were then stained with Hoechst and antibodies (Abs) against intracellular NSs and imaged by super-resolution stimulated emission depletion (STED) microscopy. 3D-reconstruction of STED Z-stacks was achieved with IMARIS software and shows nuclear NSs filaments in red and the nuclei of infected cells in blue. Experiments were repeated independently three times with similar results. Scale bar, 5 µm. c High magnification STED microscopy image of a nuclear NSs filament (red). Results are representative of three independent experiments. Scale bar, 1 µm. d Transmission electron microscopy (TEM) of nuclear NSs fibrillary aggregates. Human HeLa cells were infected with RVFV (MOI ~5) and examined in thin sections 6 h pi. The black dashed line outlines one NSs fibrillary aggregate. Images are representative of five independent experiments. Scale bar, 1 µm. e High magnification TEM image of nuclear NSs filaments in HeLa cells. Note that filaments are bundles composed of many nonbranched, roughly parallel individual fibrils. Results are representative of five independent experiments. Scale bar, 200 nm. f Correlative light and electron microscopy (CLEM) images of nuclear NSs fibrillary aggregates. Vero cells were exposed to RVFV (MOI ~5) and examined in thin sections 16 h pi after immunofluorescence staining against NSs (red). The EM picture shown here was stitched from seven single electron micrographs at 12,500-fold magnification. Experiments were repeated independently three times with similar results. Scale bar, 2 µm. g Electron micrographs and STED images of infected Vero cells were analyzed for the width of nuclear NSs filaments. n = 19, 30, and 7 cells and n = 11, 7, and 10 cells were examined by TEM and STED microscopy at 4, 8, and 16 h pi, respectively. Center line, median; box limits, upper and lower quartiles; whiskers, 1.5× interquartile range; points, outliers. h Vero cells were exposed to RVFV for up to 20 h and immuno-stained against NSs prior STED microscopy analysis. Images were quantified with the deep learning-based ilastik software. The length of NSs filaments was measured as described in Supplementary Fig. 2 . n = 19, 30, 11, and 5 cells were examined at 5, 8, 16, and 20 h pi, respectively. Center line, median; box limits, upper and lower quartiles; whiskers, 1.5× interquartile range; points, outliers.
    Figure Legend Snippet: Nuclear NSs filaments are bundles of many individual thin fibrils. a Human U-87 MG cells were exposed to Rift Valley fever virus strain ZH548 (RVFV) or its counterpart devoid of the NSs sequence (RVFV ΔNSs), both at MOI ~5. Infected cells were imaged by confocal microscopy after labeling of nuclei with Hoechst (blue) and immunofluorescence staining of the RVFV proteins NSs (green) and N (red). Images are representative of three independent experiments. Scale bar, 5 µm; pi post-infection. b Green monkey Vero cells were infected with RVFV at a MOI of 5 for 16 h. Samples were then stained with Hoechst and antibodies (Abs) against intracellular NSs and imaged by super-resolution stimulated emission depletion (STED) microscopy. 3D-reconstruction of STED Z-stacks was achieved with IMARIS software and shows nuclear NSs filaments in red and the nuclei of infected cells in blue. Experiments were repeated independently three times with similar results. Scale bar, 5 µm. c High magnification STED microscopy image of a nuclear NSs filament (red). Results are representative of three independent experiments. Scale bar, 1 µm. d Transmission electron microscopy (TEM) of nuclear NSs fibrillary aggregates. Human HeLa cells were infected with RVFV (MOI ~5) and examined in thin sections 6 h pi. The black dashed line outlines one NSs fibrillary aggregate. Images are representative of five independent experiments. Scale bar, 1 µm. e High magnification TEM image of nuclear NSs filaments in HeLa cells. Note that filaments are bundles composed of many nonbranched, roughly parallel individual fibrils. Results are representative of five independent experiments. Scale bar, 200 nm. f Correlative light and electron microscopy (CLEM) images of nuclear NSs fibrillary aggregates. Vero cells were exposed to RVFV (MOI ~5) and examined in thin sections 16 h pi after immunofluorescence staining against NSs (red). The EM picture shown here was stitched from seven single electron micrographs at 12,500-fold magnification. Experiments were repeated independently three times with similar results. Scale bar, 2 µm. g Electron micrographs and STED images of infected Vero cells were analyzed for the width of nuclear NSs filaments. n = 19, 30, and 7 cells and n = 11, 7, and 10 cells were examined by TEM and STED microscopy at 4, 8, and 16 h pi, respectively. Center line, median; box limits, upper and lower quartiles; whiskers, 1.5× interquartile range; points, outliers. h Vero cells were exposed to RVFV for up to 20 h and immuno-stained against NSs prior STED microscopy analysis. Images were quantified with the deep learning-based ilastik software. The length of NSs filaments was measured as described in Supplementary Fig. 2 . n = 19, 30, 11, and 5 cells were examined at 5, 8, 16, and 20 h pi, respectively. Center line, median; box limits, upper and lower quartiles; whiskers, 1.5× interquartile range; points, outliers.

    Techniques Used: Sequencing, Infection, Confocal Microscopy, Labeling, Immunofluorescence, Staining, Microscopy, Software, Transmission Assay, Electron Microscopy, Transmission Electron Microscopy

    33) Product Images from "Fluorescent protein-tagged Vpr dissociates from HIV-1 core after viral fusion and rapidly enters the cell nucleus"

    Article Title: Fluorescent protein-tagged Vpr dissociates from HIV-1 core after viral fusion and rapidly enters the cell nucleus

    Journal: Retrovirology

    doi: 10.1186/s12977-015-0215-z

    Nuclear accumulation of YFP-Vpr correlates with virus input. a , b Varying dilutions of YFP-Vpr/Gag-imCherry labeled ASLVpp were pre-bound to CV-1/TVA950 ( a ) or A549/TVA950 ( b ) cells in the cold followed by incubation at 37 °C for 2 h in the presence of 20 μΜ MG132. The nuclear YFP-Vpr was measured in fixed samples in the absence ( filled circles ) or in the presence ( open circles ) of 50 μg/ml R99 peptide. Data are means and SEM from four image fields. Linear regression lines are shown. c ASLVpp fusion-mediated nuclear accumulation of YFP-Vpr in CV-1/TVA950 cells in the absence ( filled circles ) or in the presence ( gray circles ) of 20 μΜ MG132. Data represent the ratio of nuclear YFP-Vpr to the signal prior to initiation of fusion. d The fusion efficiency of ASLVpp/YFP-Vpr/Gag-imCherry with CV-1/TVA950 or A549/TVA950 cells, as determined by single particle tracking. Bars are means and SEM from three experiments each, with total number of dual-labeled viral particles indicated. e Correlation between the fraction of viral YFP-Vpr accumulated in the nucleus after 2 h at 37 °C (see the legend to a ) and the slope of viral fusion (BlaM signal) vs the viral p24 input (see Additional file 3 : Figure S6). Every point represents a distinct ASLVpp or VSVpp preparation. f ASLVpp ( lanes 1, 2 ) and VSVpp ( lanes 3, 4 ) were produced using the wild-type HIV-1 R9ΔEnv backbone and co-labeled with YFP-Vpr (and BlaM-Vpr to enable measurements of virus-cell fusion shown in Fig. 5a). The viruses either contained ( odd lanes ) or lacked ( even lanes ) Gag-imCherry. Equal amounts of p24 from virus preparations were subjected to SDS-PAGE and blotted for p24 (HIV IG antibody) or YFP-Vpr (GFP antibody). The loading order: 1 ASLVpp/YFP-Vpr, 2 ASLVpp/YFP-Vpr/Gag-imCherry, 3 VSVpp/YFP-Vpr and 4 VSVpp/YFP-Vpr/Gag-imCherry. The numbers are the respective fractions (%) of viral YFP-Vpr that entered the nucleus in live cell experiments within 2 h
    Figure Legend Snippet: Nuclear accumulation of YFP-Vpr correlates with virus input. a , b Varying dilutions of YFP-Vpr/Gag-imCherry labeled ASLVpp were pre-bound to CV-1/TVA950 ( a ) or A549/TVA950 ( b ) cells in the cold followed by incubation at 37 °C for 2 h in the presence of 20 μΜ MG132. The nuclear YFP-Vpr was measured in fixed samples in the absence ( filled circles ) or in the presence ( open circles ) of 50 μg/ml R99 peptide. Data are means and SEM from four image fields. Linear regression lines are shown. c ASLVpp fusion-mediated nuclear accumulation of YFP-Vpr in CV-1/TVA950 cells in the absence ( filled circles ) or in the presence ( gray circles ) of 20 μΜ MG132. Data represent the ratio of nuclear YFP-Vpr to the signal prior to initiation of fusion. d The fusion efficiency of ASLVpp/YFP-Vpr/Gag-imCherry with CV-1/TVA950 or A549/TVA950 cells, as determined by single particle tracking. Bars are means and SEM from three experiments each, with total number of dual-labeled viral particles indicated. e Correlation between the fraction of viral YFP-Vpr accumulated in the nucleus after 2 h at 37 °C (see the legend to a ) and the slope of viral fusion (BlaM signal) vs the viral p24 input (see Additional file 3 : Figure S6). Every point represents a distinct ASLVpp or VSVpp preparation. f ASLVpp ( lanes 1, 2 ) and VSVpp ( lanes 3, 4 ) were produced using the wild-type HIV-1 R9ΔEnv backbone and co-labeled with YFP-Vpr (and BlaM-Vpr to enable measurements of virus-cell fusion shown in Fig. 5a). The viruses either contained ( odd lanes ) or lacked ( even lanes ) Gag-imCherry. Equal amounts of p24 from virus preparations were subjected to SDS-PAGE and blotted for p24 (HIV IG antibody) or YFP-Vpr (GFP antibody). The loading order: 1 ASLVpp/YFP-Vpr, 2 ASLVpp/YFP-Vpr/Gag-imCherry, 3 VSVpp/YFP-Vpr and 4 VSVpp/YFP-Vpr/Gag-imCherry. The numbers are the respective fractions (%) of viral YFP-Vpr that entered the nucleus in live cell experiments within 2 h

    Techniques Used: Labeling, Incubation, Single-particle Tracking, Produced, SDS Page

    YFP-Vpr shedding is rate-limiting for nuclear entry and is not modulated by capsid stability. a Nuclear GFP-Vpr and Hoechst fluorescence before and after GFP photobleaching. b Fluorescence recovery after photobleaching. Circles are normalized means and SEM of 10 nuclei. Inset The GFP-Vpr signal recovery after photobleaching ( circles ), the line is a double-exponential fit to the data. c Kinetics of Vpr nuclear accumulation in CV-1/TVA950 and A549/TVA950 cells in the presence or absence of 50 μg/ml of R99 peptide. d Kinetics of single ASLVpp fusion and YFP-Vpr shedding in CV-1- or A549-derived cells measured as the time-point of mCherry disappearance from dual-labeled ASLVpp and complete loss of YFP-Vpr, respectively. Circles represent normalized cumulative plots for signal disappearance from ASLVpp. Lifetimes of post-fusion cores were measured as the difference in disappearance times of mCherry and YFP signals for the same particle. e Synchronized fusion of ASLV from endosomes. ASLVpp was allowed to enter CV-1-derived cells for 45 min at 37 °C in the presence of 70 mM NH 4 Cl. Viral fusion was initiated by replacing NH 4 Cl with imaging buffer, and the kinetics of fusion (release of mCherry) and loss of YFP-Vpr was measured ( left axis ). The corresponding appearance of YFP-Vpr in the nucleus in the same imaging field was determined as a fold-increase over that prior to initiation of synchronous fusion from endosomes ( open circles , right axis ). f CV-1/TVA950 cells inoculated with YFP-Vpr-labeled VSVpp containing either the wild-type (WT) HIV-1 capsid (R9 backbone) or one of the two capsid mutants, K203A (destabilizing) and 5Mut (stabilizing). The amount of cell-bound viruses was equalized based on cell-associated YFP-Vpr fluorescence and viruses were allowed to fuse for 2 h at 37 °C in the presence of 20 μΜ MG132. The nuclear YFP-Vpr signal was measured at indicated time intervals and normalized to the value at 2 h. Data-points are means and SEM from four image fields each
    Figure Legend Snippet: YFP-Vpr shedding is rate-limiting for nuclear entry and is not modulated by capsid stability. a Nuclear GFP-Vpr and Hoechst fluorescence before and after GFP photobleaching. b Fluorescence recovery after photobleaching. Circles are normalized means and SEM of 10 nuclei. Inset The GFP-Vpr signal recovery after photobleaching ( circles ), the line is a double-exponential fit to the data. c Kinetics of Vpr nuclear accumulation in CV-1/TVA950 and A549/TVA950 cells in the presence or absence of 50 μg/ml of R99 peptide. d Kinetics of single ASLVpp fusion and YFP-Vpr shedding in CV-1- or A549-derived cells measured as the time-point of mCherry disappearance from dual-labeled ASLVpp and complete loss of YFP-Vpr, respectively. Circles represent normalized cumulative plots for signal disappearance from ASLVpp. Lifetimes of post-fusion cores were measured as the difference in disappearance times of mCherry and YFP signals for the same particle. e Synchronized fusion of ASLV from endosomes. ASLVpp was allowed to enter CV-1-derived cells for 45 min at 37 °C in the presence of 70 mM NH 4 Cl. Viral fusion was initiated by replacing NH 4 Cl with imaging buffer, and the kinetics of fusion (release of mCherry) and loss of YFP-Vpr was measured ( left axis ). The corresponding appearance of YFP-Vpr in the nucleus in the same imaging field was determined as a fold-increase over that prior to initiation of synchronous fusion from endosomes ( open circles , right axis ). f CV-1/TVA950 cells inoculated with YFP-Vpr-labeled VSVpp containing either the wild-type (WT) HIV-1 capsid (R9 backbone) or one of the two capsid mutants, K203A (destabilizing) and 5Mut (stabilizing). The amount of cell-bound viruses was equalized based on cell-associated YFP-Vpr fluorescence and viruses were allowed to fuse for 2 h at 37 °C in the presence of 20 μΜ MG132. The nuclear YFP-Vpr signal was measured at indicated time intervals and normalized to the value at 2 h. Data-points are means and SEM from four image fields each

    Techniques Used: Fluorescence, Derivative Assay, Labeling, Imaging

    Post-fusion decay of HIV-1 YFP-Vpr signal. a , d ASLVpp co-labeled with the core-associated YFP-Vpr ( green ) and a releasable content marker Gag-imCherry ( dark red ) were pre-bound in the cold to CV-1/TVA950 ( a – c , g ) or A549/TVA950 ( d – f ) cells expressing the ASLV receptor TVA950. Entry was initiated by introducing warm buffer, and cells were maintained at 37 °C for 45 min and imaged every 3–5 s. Fusing viruses were detected by the near-instantaneous disappearance of mCherry from double-labeled particles (marked by white circles in a and d ). White dashed lines show the boundaries of cell nuclei. b , c Fluorescence intensity profiles (total fluorescence of YFP-Vpr and Gag-imCherry) obtained by single ASLVpp tracking in CV-1-derived cells. e , f Fluorescence intensity profiles for YFP-Vpr and Gag-imCherry obtained by single ASLVpp tracking in an A549-derived cell. g An example of YFP-Vpr and Gag-imCherry signals from a non-fusing particle selected from an experiment carried out in the presence of the ASLV fusion inhibitor R99 (50 μg/ml). Black dashed lines outline different YFP decay profiles occurring without ( c , e ) and with a lag ( b , f ) after the release of mCherry. Here and in Fig. 2 , the abrupt ending of fluorescence traces occurs due to the inability to track faint YFP/GFP-Vpr puncta using particle tracking software, as the signal approaches the background level
    Figure Legend Snippet: Post-fusion decay of HIV-1 YFP-Vpr signal. a , d ASLVpp co-labeled with the core-associated YFP-Vpr ( green ) and a releasable content marker Gag-imCherry ( dark red ) were pre-bound in the cold to CV-1/TVA950 ( a – c , g ) or A549/TVA950 ( d – f ) cells expressing the ASLV receptor TVA950. Entry was initiated by introducing warm buffer, and cells were maintained at 37 °C for 45 min and imaged every 3–5 s. Fusing viruses were detected by the near-instantaneous disappearance of mCherry from double-labeled particles (marked by white circles in a and d ). White dashed lines show the boundaries of cell nuclei. b , c Fluorescence intensity profiles (total fluorescence of YFP-Vpr and Gag-imCherry) obtained by single ASLVpp tracking in CV-1-derived cells. e , f Fluorescence intensity profiles for YFP-Vpr and Gag-imCherry obtained by single ASLVpp tracking in an A549-derived cell. g An example of YFP-Vpr and Gag-imCherry signals from a non-fusing particle selected from an experiment carried out in the presence of the ASLV fusion inhibitor R99 (50 μg/ml). Black dashed lines outline different YFP decay profiles occurring without ( c , e ) and with a lag ( b , f ) after the release of mCherry. Here and in Fig. 2 , the abrupt ending of fluorescence traces occurs due to the inability to track faint YFP/GFP-Vpr puncta using particle tracking software, as the signal approaches the background level

    Techniques Used: Labeling, Marker, Expressing, Fluorescence, Derivative Assay, Software

    Loss of YFP-Vpr after viral fusion mediated by HXB2 envelope glycoprotein. a Snapshots of entry and fusion of an HXB2 Env-pseudotyped particle co-labeled with YFP-Vpr ( green ) and Gag-imCherry ( red ). Viruses were pre-bound to CV-1 cells expressing CD4 and CXCR4 and incubated for 1 h at 37 °C to allow fusion, which was manifested by an abrupt loss of the mCherry marker. Post-fusion decay of the YFP-Vpr signal is evident from the lowest image panel. b Single particle tracking of the virus in a , showing a virtually instantaneous loss of mCherry ( dark red ) followed by a gradual decay of the YFP signal ( green ). c – e Examples of HXB2 pp fusion (release of mCherry) with subsequent reduction in the YFP-Vpr fluorescence for pseudoviruses produced using pR8ΔEnv ( c ) and psPAX2 ( d , e ) vectors
    Figure Legend Snippet: Loss of YFP-Vpr after viral fusion mediated by HXB2 envelope glycoprotein. a Snapshots of entry and fusion of an HXB2 Env-pseudotyped particle co-labeled with YFP-Vpr ( green ) and Gag-imCherry ( red ). Viruses were pre-bound to CV-1 cells expressing CD4 and CXCR4 and incubated for 1 h at 37 °C to allow fusion, which was manifested by an abrupt loss of the mCherry marker. Post-fusion decay of the YFP-Vpr signal is evident from the lowest image panel. b Single particle tracking of the virus in a , showing a virtually instantaneous loss of mCherry ( dark red ) followed by a gradual decay of the YFP signal ( green ). c – e Examples of HXB2 pp fusion (release of mCherry) with subsequent reduction in the YFP-Vpr fluorescence for pseudoviruses produced using pR8ΔEnv ( c ) and psPAX2 ( d , e ) vectors

    Techniques Used: Labeling, Expressing, Incubation, Marker, Single-particle Tracking, Fluorescence, Produced

    Nuclear accumulation of YFP-Vpr upon co-incubation of viruses with target cells. a ASLVpp labeled with YFP-Vpr ( green ) and Gag-imCherry ( red ) were pre-bound to CV-1 cells expressing TVA950 in the cold ( left panels ), and virus entry was initiated by shifting to 37 °C. At 45 min post-initiation, 70 mM of NH 4 Cl was added to block fusion and fully recover the YFP fluorescence in acidic endosomes ( middle panels ). Virus-cell incubation in the presence of fusion inhibitory R99 peptide (50 μg/ml) abrogates nuclear accumulation of YFP-Vpr, but not virus uptake ( right panels ). The top panels are three-color (Hoechst/YFP-Vpr/Gag-imCherry) images, while the bottom panels show only the YFP-Vpr and Gag-imCherry channels for clarity. The left and middle panels are the same image field at different time points, while the right panels are from a different experiment carried out in the presence of R99. White lines are drawn through the nuclei to generate respective intensity profiles shown in d . Inset in the lower middle panel shows the enlarged boxed area. b High-resolution confocal image of an optical slice through the middle of the CV-1 cell nucleus stained with Hoechst-33342 after incubation with MOI of ~0.05 of ASLVpp co-labeled with YFP-Vpr and Gag-imCherry. c Line histograms through nuclei corresponding to images in ( a ) depict the degree of spatial overlap of YFP-Vpr ( green ), mCherry ( red ) and Hoechst ( blue ) signals before raising the temperature and after 45 min at 37 °C in the absence or in the presence of R99 peptide. d Images of YFP-Vpr/Gag-imCherry labeled HXB2 pp particles pre-bound to CV-1-derived target cells before ( left ) and after ( middle ) incubation at 37 °C for 3 h. Parallel samples ( right ) were incubated for 3 h in the presence of 5 μM of HIV-1 fusion inhibitor C52L. Three-color images ( upper panels ) and two-color images ( lower panels ) are shown for the ease of identification of the YFP-Vpr signal within the Hoechst-stained nuclei ( blue )
    Figure Legend Snippet: Nuclear accumulation of YFP-Vpr upon co-incubation of viruses with target cells. a ASLVpp labeled with YFP-Vpr ( green ) and Gag-imCherry ( red ) were pre-bound to CV-1 cells expressing TVA950 in the cold ( left panels ), and virus entry was initiated by shifting to 37 °C. At 45 min post-initiation, 70 mM of NH 4 Cl was added to block fusion and fully recover the YFP fluorescence in acidic endosomes ( middle panels ). Virus-cell incubation in the presence of fusion inhibitory R99 peptide (50 μg/ml) abrogates nuclear accumulation of YFP-Vpr, but not virus uptake ( right panels ). The top panels are three-color (Hoechst/YFP-Vpr/Gag-imCherry) images, while the bottom panels show only the YFP-Vpr and Gag-imCherry channels for clarity. The left and middle panels are the same image field at different time points, while the right panels are from a different experiment carried out in the presence of R99. White lines are drawn through the nuclei to generate respective intensity profiles shown in d . Inset in the lower middle panel shows the enlarged boxed area. b High-resolution confocal image of an optical slice through the middle of the CV-1 cell nucleus stained with Hoechst-33342 after incubation with MOI of ~0.05 of ASLVpp co-labeled with YFP-Vpr and Gag-imCherry. c Line histograms through nuclei corresponding to images in ( a ) depict the degree of spatial overlap of YFP-Vpr ( green ), mCherry ( red ) and Hoechst ( blue ) signals before raising the temperature and after 45 min at 37 °C in the absence or in the presence of R99 peptide. d Images of YFP-Vpr/Gag-imCherry labeled HXB2 pp particles pre-bound to CV-1-derived target cells before ( left ) and after ( middle ) incubation at 37 °C for 3 h. Parallel samples ( right ) were incubated for 3 h in the presence of 5 μM of HIV-1 fusion inhibitor C52L. Three-color images ( upper panels ) and two-color images ( lower panels ) are shown for the ease of identification of the YFP-Vpr signal within the Hoechst-stained nuclei ( blue )

    Techniques Used: Incubation, Labeling, Expressing, Blocking Assay, Fluorescence, Staining, Derivative Assay

    Single VSV-G pseudovirus fusion results in loss of GFP-Vpr from viral core. VSVpp co-labeled with Gag-imCherry (content marker, red ) and GFP-Vpr (core marker, green ) were pre-bound to CV-1 cells and allowed to fuse at 37 °C in Fluorobrite DMEM medium under 5 % CO 2 . a Images of mCherry release from single virus followed by gradual loss of the GFP-Vpr signal. b Dark red and green traces show sum fluorescence of mCherry and GFP channels, respectively, obtain by tracking the virus shown in a . For comparison, fluorescence intensities of mCherry and GFP for a non-fusing particle are shown ( beige and cyan traces, respectively). c Single virus tracking results of another fusing VSVpp. Occasional spikes in fluorescence (for example, at the 38 min time point) are due to a transient overlap of the particle of interest with either another particle or with cell’s autofluorescent features
    Figure Legend Snippet: Single VSV-G pseudovirus fusion results in loss of GFP-Vpr from viral core. VSVpp co-labeled with Gag-imCherry (content marker, red ) and GFP-Vpr (core marker, green ) were pre-bound to CV-1 cells and allowed to fuse at 37 °C in Fluorobrite DMEM medium under 5 % CO 2 . a Images of mCherry release from single virus followed by gradual loss of the GFP-Vpr signal. b Dark red and green traces show sum fluorescence of mCherry and GFP channels, respectively, obtain by tracking the virus shown in a . For comparison, fluorescence intensities of mCherry and GFP for a non-fusing particle are shown ( beige and cyan traces, respectively). c Single virus tracking results of another fusing VSVpp. Occasional spikes in fluorescence (for example, at the 38 min time point) are due to a transient overlap of the particle of interest with either another particle or with cell’s autofluorescent features

    Techniques Used: Labeling, Marker, Fluorescence

    34) Product Images from "Ajuba receptor mediates the internalization of tumor-secreted GRP78 into macrophages through different endocytosis pathways"

    Article Title: Ajuba receptor mediates the internalization of tumor-secreted GRP78 into macrophages through different endocytosis pathways

    Journal: Oncotarget

    doi: 10.18632/oncotarget.24090

    Phagocytosis is not the only way for secreted GRP78 to enter into macrophages ( A, D, G, J ) Location of GRP78 in FHC, COS-7, DLD1, HeLa cells treated with 40 nM FITC-GRP78 at different time points. Corresponding images were superimposed to determine the location of GRP78. Scale bars represent 6 μm. ( B, E, H, K ) Concentration of GRP78 protein in the above cells. As described in (A, D, G, J), experiments were repeated three times, and 250 cell per time point in each experiment were scored in the quantification analysis using Image J software. ( C, F, I, L ) Levels of internalized protein. As described in (A, D, G, J), Western blot data were superimposed to determine the levels of internalized protein. Mouse anti-GAPDH antibodies was used as a loading control.
    Figure Legend Snippet: Phagocytosis is not the only way for secreted GRP78 to enter into macrophages ( A, D, G, J ) Location of GRP78 in FHC, COS-7, DLD1, HeLa cells treated with 40 nM FITC-GRP78 at different time points. Corresponding images were superimposed to determine the location of GRP78. Scale bars represent 6 μm. ( B, E, H, K ) Concentration of GRP78 protein in the above cells. As described in (A, D, G, J), experiments were repeated three times, and 250 cell per time point in each experiment were scored in the quantification analysis using Image J software. ( C, F, I, L ) Levels of internalized protein. As described in (A, D, G, J), Western blot data were superimposed to determine the levels of internalized protein. Mouse anti-GAPDH antibodies was used as a loading control.

    Techniques Used: Concentration Assay, Software, Western Blot

    35) Product Images from "Deletion of the Vaccinia Virus B1 Kinase Reveals Essential Functions of This Enzyme Complemented Partly by the Homologous Cellular Kinase VRK2"

    Article Title: Deletion of the Vaccinia Virus B1 Kinase Reveals Essential Functions of This Enzyme Complemented Partly by the Homologous Cellular Kinase VRK2

    Journal: Journal of Virology

    doi: 10.1128/JVI.00635-17

    Growth of vvΔB1 in multiple cell types. One-step infections were done at an MOI of 3 for WT (black) or vvΔB1 (red) virus on BSC40, CV1, L929, or HeLa cells incubated at 37°C. Cells and virus were harvested at 24 hpi. (A) DNA accumulation for BSC40, CV1, and L929 cells. The value for the WT/BSC40 sample was set to 1. (B) Viral yield for infections in BSC40, CV1, and L929 cells. Virus titrations were completed on B1myc-expressing CV1 cells. (C and D) HeLa cell DNA accumulation (C) and viral yield (D) assays were conducted separately from those for other cell lines, using an identical protocol. The value for the WT/HeLa sample was set to 1. Titers of viral harvest were determined on CV1-B1myc cells. Standard deviation is denoted by error bars, and P values are indicated as follows: †,
    Figure Legend Snippet: Growth of vvΔB1 in multiple cell types. One-step infections were done at an MOI of 3 for WT (black) or vvΔB1 (red) virus on BSC40, CV1, L929, or HeLa cells incubated at 37°C. Cells and virus were harvested at 24 hpi. (A) DNA accumulation for BSC40, CV1, and L929 cells. The value for the WT/BSC40 sample was set to 1. (B) Viral yield for infections in BSC40, CV1, and L929 cells. Virus titrations were completed on B1myc-expressing CV1 cells. (C and D) HeLa cell DNA accumulation (C) and viral yield (D) assays were conducted separately from those for other cell lines, using an identical protocol. The value for the WT/HeLa sample was set to 1. Titers of viral harvest were determined on CV1-B1myc cells. Standard deviation is denoted by error bars, and P values are indicated as follows: †,

    Techniques Used: Incubation, Expressing, Standard Deviation

    36) Product Images from "Pro-hormone Secretogranin II Regulates Dense Core Secretory Granule Biogenesis in Catecholaminergic Cells *"

    Article Title: Pro-hormone Secretogranin II Regulates Dense Core Secretory Granule Biogenesis in Catecholaminergic Cells *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.064196

    Secretagogue-stimulated exocytosis of SgII in COS-7 and A35C cells. A and B , regulated secretion of EAP fusion proteins. COS-7 cells ( A ) and A35C cells ( B ) expressing SIG-EAP or SgII-EAP were exposed for 30 min to DMEM alone (−) or DMEM supplemented with the indicated concentration of A23187 (+). EAP type secretion was assayed as described under “Experimental Procedures.” Release of EAP is expressed either as % of EAP activity secretion ( A, left ) or relative to enzymatic activity released in the absence of secretagogue ( A, right, and B ). Values are given as the mean ± S.E. of triplicate determinations. Results from one of at least three independent experiments are shown. †, p > 0.05; ***, p
    Figure Legend Snippet: Secretagogue-stimulated exocytosis of SgII in COS-7 and A35C cells. A and B , regulated secretion of EAP fusion proteins. COS-7 cells ( A ) and A35C cells ( B ) expressing SIG-EAP or SgII-EAP were exposed for 30 min to DMEM alone (−) or DMEM supplemented with the indicated concentration of A23187 (+). EAP type secretion was assayed as described under “Experimental Procedures.” Release of EAP is expressed either as % of EAP activity secretion ( A, left ) or relative to enzymatic activity released in the absence of secretagogue ( A, right, and B ). Values are given as the mean ± S.E. of triplicate determinations. Results from one of at least three independent experiments are shown. †, p > 0.05; ***, p

    Techniques Used: Expressing, Concentration Assay, Activity Assay

    37) Product Images from "HIF and HOIL-1L–mediated PKCζ degradation stabilizes plasma membrane Na,K-ATPase to protect against hypoxia-induced lung injury"

    Article Title: HIF and HOIL-1L–mediated PKCζ degradation stabilizes plasma membrane Na,K-ATPase to protect against hypoxia-induced lung injury

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

    doi: 10.1073/pnas.1713563114

    PKCζ degradation is initiated at the cell plasma membrane. ( A ) COS-7 cells were transfected with FLAG-WT-PKCζ (WT) or FLAG-T410A-PKCζ (T410A) and exposed to 1.5% O 2 . E-cadherin was used as loading control for membrane fraction, and β-actin was used as a marker of cytosolic proteins. Representative immunoblots from four independent experiments are shown. ( B ) Isolated plasma membrane proteins from rat ATII cells exposed to normoxia or 1.5% O 2 were immunoblotted using an anti-PKCζ antibody. E-cadherin was used as loading control ( n = 3). Statistical significance was calculated using one-way ANOVA and the Dunnett’s multiple comparisons test (** P
    Figure Legend Snippet: PKCζ degradation is initiated at the cell plasma membrane. ( A ) COS-7 cells were transfected with FLAG-WT-PKCζ (WT) or FLAG-T410A-PKCζ (T410A) and exposed to 1.5% O 2 . E-cadherin was used as loading control for membrane fraction, and β-actin was used as a marker of cytosolic proteins. Representative immunoblots from four independent experiments are shown. ( B ) Isolated plasma membrane proteins from rat ATII cells exposed to normoxia or 1.5% O 2 were immunoblotted using an anti-PKCζ antibody. E-cadherin was used as loading control ( n = 3). Statistical significance was calculated using one-way ANOVA and the Dunnett’s multiple comparisons test (** P

    Techniques Used: Transfection, Marker, Western Blot, Isolation

    PKCζ phosphorylation is required for its ubiquitylation and degradation. Cells were exposed to normoxia or 1.5% O 2 for up to 24 h. ( A ) COS-7 cells were transfected with FLAG-PKCζ-WT, and 4 h after treatment cells were lysed and proteins were immunoprecipitated with anti-FLAG antibody. Densitometry quantification of immunoblots of phospho-PKCζ (pPKCζ) in relation to FLAG is shown ( n = 3). ( B ) Rat ATII cells were infected with null adenovirus (Null) or HA-tagged adenovirus carrying a dominant-negative, kinase-dead (K45R) variant of AMPK (DN-AMPK) after 24 h of normoxia or 1.5% O 2 . PKCζ expression was evaluated by Western blot; β-actin was used as a loading control ( n = 3). Statistical significance was calculated for A and B using one-way ANOVA and the Tukey multiple comparisons test (* P
    Figure Legend Snippet: PKCζ phosphorylation is required for its ubiquitylation and degradation. Cells were exposed to normoxia or 1.5% O 2 for up to 24 h. ( A ) COS-7 cells were transfected with FLAG-PKCζ-WT, and 4 h after treatment cells were lysed and proteins were immunoprecipitated with anti-FLAG antibody. Densitometry quantification of immunoblots of phospho-PKCζ (pPKCζ) in relation to FLAG is shown ( n = 3). ( B ) Rat ATII cells were infected with null adenovirus (Null) or HA-tagged adenovirus carrying a dominant-negative, kinase-dead (K45R) variant of AMPK (DN-AMPK) after 24 h of normoxia or 1.5% O 2 . PKCζ expression was evaluated by Western blot; β-actin was used as a loading control ( n = 3). Statistical significance was calculated for A and B using one-way ANOVA and the Tukey multiple comparisons test (* P

    Techniques Used: Transfection, Immunoprecipitation, Western Blot, Infection, Dominant Negative Mutation, Variant Assay, Expressing

    38) Product Images from "Palmitoylation of the Rous Sarcoma Virus Transmembrane Glycoprotein Is Required for Protein Stability and Virus Infectivity"

    Article Title: Palmitoylation of the Rous Sarcoma Virus Transmembrane Glycoprotein Is Required for Protein Stability and Virus Infectivity

    Journal: Journal of Virology

    doi: 10.1128/JVI.75.23.11544-11554.2001

    Labeling of RSV glycoproteins with [ 3 H]palmitic acid. Wild-type and mutant glycoproteins expressed in CV-1 cells after infection with the recombinant SV40 vector were labeled for 4 h with [ 3 H]palmitic acid. Immunoprecipitates were separated on SDS–12% PAGE and then analyzed by fluorography. Lane 1, wild type (wt); lane 2, C164G; lane 3, C167G; lane 4, C164G/C167G; lane 5, mock infected.
    Figure Legend Snippet: Labeling of RSV glycoproteins with [ 3 H]palmitic acid. Wild-type and mutant glycoproteins expressed in CV-1 cells after infection with the recombinant SV40 vector were labeled for 4 h with [ 3 H]palmitic acid. Immunoprecipitates were separated on SDS–12% PAGE and then analyzed by fluorography. Lane 1, wild type (wt); lane 2, C164G; lane 3, C167G; lane 4, C164G/C167G; lane 5, mock infected.

    Techniques Used: Labeling, Mutagenesis, Infection, Recombinant, Plasmid Preparation, Polyacrylamide Gel Electrophoresis

    Indirect immunofluorescence of wild-type (wt) and palmitoylation mutant glycoproteins in CV-1 cells reveals different steady-state distributions. The intracellular distributions of wild-type and mutant env gene products were probed after acetone fixation and permeabilization of recombinant-SV40-infected CV-1 cells grown on glass coverslips using rb-anti-RSV-TM-Cpep (Whole Cell IF). Steady-state surface expression of wild-type and mutant env ).
    Figure Legend Snippet: Indirect immunofluorescence of wild-type (wt) and palmitoylation mutant glycoproteins in CV-1 cells reveals different steady-state distributions. The intracellular distributions of wild-type and mutant env gene products were probed after acetone fixation and permeabilization of recombinant-SV40-infected CV-1 cells grown on glass coverslips using rb-anti-RSV-TM-Cpep (Whole Cell IF). Steady-state surface expression of wild-type and mutant env ).

    Techniques Used: Immunofluorescence, Mutagenesis, Recombinant, Infection, Expressing

    39) Product Images from "Amphiastral Mitotic Spindle Assembly in Vertebrate Cells Lacking Centrosomes"

    Article Title: Amphiastral Mitotic Spindle Assembly in Vertebrate Cells Lacking Centrosomes

    Journal: Current biology : CB

    doi: 10.1016/j.cub.2011.02.049

    Microsurgical removal of the centrosome from BSC-1 cells Aa . The centrosome is identified by a mass of granules adjacent to the nucleus (arrow). Ab–Ae. The needle is brought down between the nucleus and the centrosome. Af. The nuclear containing karyoplast (Karyo or KP) is completely separated from the centrosome containing cytoplast (cyto or CP). B. MT network reformation in the KP/CP pair. Ba–c. Two cells following microsurgery. The first is a mock-cut, where the centrosome is displaced from the nucleus by the needle, but the CP is not severed from the KP. The second cell is cut by the needle, resulting in a KP/CP pair. The KP is surrounded by a white outline. Bc. Fluorescence imaging: the mock-cut cell and the KP both lack a perinuclear MT focus, whereas the CP has a radial MT array. Bd–bf. The radial MTs reforms in the mock-cut cell (arrow in e ) and KP (arrow in Bf ). C. aMTOC formation in the karyoplast. Ca–Cd. The KP/CP flatten out over the first 1½ hours. Cd ′ -Cd ‴’. Same cell immunolabeling of d . The MTs in the KP ( d ′) do not have a central focus; the control cell does (arrowhead). The centrin-2 positive centrioles ( Cd ″) and γ-tubulin positive pericentriolar material ( Cd ‴) are in the CP. Ce–Ch. The KP/CP pair ~ 5 hrs after microsurgery Ch ′ -Ch ‴’. Same cell immunofluorescence of Ch . The aMTOC has reformed (arrow in Ch ′). The KP lacks centrioles and PCM; these reside in the CP. Time = Hrs:min. Bars = 10 μm.
    Figure Legend Snippet: Microsurgical removal of the centrosome from BSC-1 cells Aa . The centrosome is identified by a mass of granules adjacent to the nucleus (arrow). Ab–Ae. The needle is brought down between the nucleus and the centrosome. Af. The nuclear containing karyoplast (Karyo or KP) is completely separated from the centrosome containing cytoplast (cyto or CP). B. MT network reformation in the KP/CP pair. Ba–c. Two cells following microsurgery. The first is a mock-cut, where the centrosome is displaced from the nucleus by the needle, but the CP is not severed from the KP. The second cell is cut by the needle, resulting in a KP/CP pair. The KP is surrounded by a white outline. Bc. Fluorescence imaging: the mock-cut cell and the KP both lack a perinuclear MT focus, whereas the CP has a radial MT array. Bd–bf. The radial MTs reforms in the mock-cut cell (arrow in e ) and KP (arrow in Bf ). C. aMTOC formation in the karyoplast. Ca–Cd. The KP/CP flatten out over the first 1½ hours. Cd ′ -Cd ‴’. Same cell immunolabeling of d . The MTs in the KP ( d ′) do not have a central focus; the control cell does (arrowhead). The centrin-2 positive centrioles ( Cd ″) and γ-tubulin positive pericentriolar material ( Cd ‴) are in the CP. Ce–Ch. The KP/CP pair ~ 5 hrs after microsurgery Ch ′ -Ch ‴’. Same cell immunofluorescence of Ch . The aMTOC has reformed (arrow in Ch ′). The KP lacks centrioles and PCM; these reside in the CP. Time = Hrs:min. Bars = 10 μm.

    Techniques Used: Fluorescence, Imaging, Immunolabeling, Immunofluorescence

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    ATCC african green monkey kidney ma104 cells
    RIG-I is down-regulated by NSP1 at the protein level but is proteasome-independent . (A, B) Western blot analysis of RIG-I down-regulation by NSP1. 293FT cells were transfected with increased amount of pEGFP-OSU NSP1 (A) or pEGFP-SA11 NSP1 (B) and plasmid encoding Myc-RIG-I. Cell extracts were prepared 48 h post-transfection. Immunoblots were probed with anti-Myc monoclonal antibody to detect RIG-I (top panel). β-actin was used as a loading control (bottom panel). (C) Transcription level of RIG-I at different time points after transfection. 293FT cells were co-transfected with SA11-NSP1 and RIG-I plasmids. At different time points after transfection, total RNA extracted from cells was subjected to RT-PCR amplification and electrophoresis for RIG-I, NSP1 and GAPDH (inner control) mRNAs. (D, E) RIG-I is degraded in rotavirus infected cells. <t>MA104</t> cells were infected with rotavirus SA11 at a m.o.i. of 0.1. Cell extracts were prepared at 0, 4, 8, 12, 24 and 36 h post-infection (p.i). RIG-I protein levels at each time point p.i. were determined by Western blot analyses using an anti-RIG-I antibody. The viral protein VP6 was used as an indicator for rotavirus infection. β-actin was used as a loading control. RIG-I, NSP1, VP6 and GAPDH mRNAs were also checked in parallel for evaluating the transcription level (E). (F, G) Effects of a proteasome inhibitor on NSP1 mediated RIG-I down-regulation. 293FT cells were transfected with Myc-RIG-I, pEGFP-OSU NSP1 (F) or pEGFP-SA11 NSP1 (G). The cells were treated with the proteasome inhibitor MG132 or an equivalent volume of DMSO as described in Methods. Lysates were prepared 36-48 h post-transfection. Immunoblots were probed with anti-Myc to detect myc-tagged RIG-I.
    African Green Monkey Kidney Ma104 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 97/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    RIG-I is down-regulated by NSP1 at the protein level but is proteasome-independent . (A, B) Western blot analysis of RIG-I down-regulation by NSP1. 293FT cells were transfected with increased amount of pEGFP-OSU NSP1 (A) or pEGFP-SA11 NSP1 (B) and plasmid encoding Myc-RIG-I. Cell extracts were prepared 48 h post-transfection. Immunoblots were probed with anti-Myc monoclonal antibody to detect RIG-I (top panel). β-actin was used as a loading control (bottom panel). (C) Transcription level of RIG-I at different time points after transfection. 293FT cells were co-transfected with SA11-NSP1 and RIG-I plasmids. At different time points after transfection, total RNA extracted from cells was subjected to RT-PCR amplification and electrophoresis for RIG-I, NSP1 and GAPDH (inner control) mRNAs. (D, E) RIG-I is degraded in rotavirus infected cells. MA104 cells were infected with rotavirus SA11 at a m.o.i. of 0.1. Cell extracts were prepared at 0, 4, 8, 12, 24 and 36 h post-infection (p.i). RIG-I protein levels at each time point p.i. were determined by Western blot analyses using an anti-RIG-I antibody. The viral protein VP6 was used as an indicator for rotavirus infection. β-actin was used as a loading control. RIG-I, NSP1, VP6 and GAPDH mRNAs were also checked in parallel for evaluating the transcription level (E). (F, G) Effects of a proteasome inhibitor on NSP1 mediated RIG-I down-regulation. 293FT cells were transfected with Myc-RIG-I, pEGFP-OSU NSP1 (F) or pEGFP-SA11 NSP1 (G). The cells were treated with the proteasome inhibitor MG132 or an equivalent volume of DMSO as described in Methods. Lysates were prepared 36-48 h post-transfection. Immunoblots were probed with anti-Myc to detect myc-tagged RIG-I.

    Journal: Virology Journal

    Article Title: Rotavirus nonstructural protein 1 antagonizes innate immune response by interacting with retinoic acid inducible gene I

    doi: 10.1186/1743-422X-8-526

    Figure Lengend Snippet: RIG-I is down-regulated by NSP1 at the protein level but is proteasome-independent . (A, B) Western blot analysis of RIG-I down-regulation by NSP1. 293FT cells were transfected with increased amount of pEGFP-OSU NSP1 (A) or pEGFP-SA11 NSP1 (B) and plasmid encoding Myc-RIG-I. Cell extracts were prepared 48 h post-transfection. Immunoblots were probed with anti-Myc monoclonal antibody to detect RIG-I (top panel). β-actin was used as a loading control (bottom panel). (C) Transcription level of RIG-I at different time points after transfection. 293FT cells were co-transfected with SA11-NSP1 and RIG-I plasmids. At different time points after transfection, total RNA extracted from cells was subjected to RT-PCR amplification and electrophoresis for RIG-I, NSP1 and GAPDH (inner control) mRNAs. (D, E) RIG-I is degraded in rotavirus infected cells. MA104 cells were infected with rotavirus SA11 at a m.o.i. of 0.1. Cell extracts were prepared at 0, 4, 8, 12, 24 and 36 h post-infection (p.i). RIG-I protein levels at each time point p.i. were determined by Western blot analyses using an anti-RIG-I antibody. The viral protein VP6 was used as an indicator for rotavirus infection. β-actin was used as a loading control. RIG-I, NSP1, VP6 and GAPDH mRNAs were also checked in parallel for evaluating the transcription level (E). (F, G) Effects of a proteasome inhibitor on NSP1 mediated RIG-I down-regulation. 293FT cells were transfected with Myc-RIG-I, pEGFP-OSU NSP1 (F) or pEGFP-SA11 NSP1 (G). The cells were treated with the proteasome inhibitor MG132 or an equivalent volume of DMSO as described in Methods. Lysates were prepared 36-48 h post-transfection. Immunoblots were probed with anti-Myc to detect myc-tagged RIG-I.

    Article Snippet: African green monkey kidney MA104 cells (ATCC, Manassas, VA) were maintained in DMEM supplemented with 5% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin.

    Techniques: Western Blot, Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Amplification, Electrophoresis, Infection

    Reciprocal trans -complementation of RIG-I- and IFN-β-deficient cells by MeV-induced fusion. RIG-I-deficient Huh7.5 or IFN-β-deficient Vero cells were infected with MeV at an MOI of 1 and cocultured 8 h later with uninfected Vero and Huh7.5

    Journal:

    Article Title: Cell-Cell Fusion Induced by Measles Virus Amplifies the Type I Interferon Response ▿Cell-Cell Fusion Induced by Measles Virus Amplifies the Type I Interferon Response ▿ †

    doi: 10.1128/JVI.00078-07

    Figure Lengend Snippet: Reciprocal trans -complementation of RIG-I- and IFN-β-deficient cells by MeV-induced fusion. RIG-I-deficient Huh7.5 or IFN-β-deficient Vero cells were infected with MeV at an MOI of 1 and cocultured 8 h later with uninfected Vero and Huh7.5

    Article Snippet: Human kidney epithelial 293T/17 cells (ATCC) expressing CD46 (293T/CD46+ ), HeLa cells, African green monkey Vero fibroblasts (ATCC), 293T/CD150+ cells , Huh7.5 cells, a subline of Huh7 cells defective in RIG-I , and human cortical thymic epithelial cells (TEC) (clone P1.4D6) from postnatal thymus ( ) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum or as described previously ( , ).

    Techniques: Infection

    Characterization of the expression of IBDV VP1 in cells infected with the rVV VT7/VP1. (A) BSC-1 cells infected with VT7/VP1 or the parental virus VT7LacOI, either treated (+) or untreated (−) with the inducer IPTG, were metabolically labelled with [ 35 S]methionine. CEF mock-infected (Mock) or infected with IBDV (IBDV) were metabolically labelled with [ 35 S]methionine at 48 h p.i. Protein samples from the different cultures were analyzed by SDS-PAGE. After electrophoresis, the gel was electroblotted onto nitrocellulose. The filter was air-dried and subjected to autoradiography. The positions of molecular weight markers are indicated (Mw). The positions of bands corresponding to the VP1 polypeptide are indicated by open arrowheads. (B) Western blot analysis of proteins expressed in cells infected with the rVV VT7/VP1 and CEF infected with IBDV. To determine the position of the VP1 polypeptide, after autoradiography, the nitrocellulose filter was rehydrated and incubated with a rabbit anti-VP1 antisera followed by the addition of horseradish peroxidase-conjugated goat anti-rabbit Ig. The signal was detected by ECL.

    Journal: Journal of Virology

    Article Title: VP1, the Putative RNA-Dependent RNA Polymerase of Infectious Bursal Disease Virus, Forms Complexes with the Capsid Protein VP3, Leading to Efficient Encapsidation into Virus-Like Particles

    doi:

    Figure Lengend Snippet: Characterization of the expression of IBDV VP1 in cells infected with the rVV VT7/VP1. (A) BSC-1 cells infected with VT7/VP1 or the parental virus VT7LacOI, either treated (+) or untreated (−) with the inducer IPTG, were metabolically labelled with [ 35 S]methionine. CEF mock-infected (Mock) or infected with IBDV (IBDV) were metabolically labelled with [ 35 S]methionine at 48 h p.i. Protein samples from the different cultures were analyzed by SDS-PAGE. After electrophoresis, the gel was electroblotted onto nitrocellulose. The filter was air-dried and subjected to autoradiography. The positions of molecular weight markers are indicated (Mw). The positions of bands corresponding to the VP1 polypeptide are indicated by open arrowheads. (B) Western blot analysis of proteins expressed in cells infected with the rVV VT7/VP1 and CEF infected with IBDV. To determine the position of the VP1 polypeptide, after autoradiography, the nitrocellulose filter was rehydrated and incubated with a rabbit anti-VP1 antisera followed by the addition of horseradish peroxidase-conjugated goat anti-rabbit Ig. The signal was detected by ECL.

    Article Snippet: The rVVs VT7/POLY, VT7/VP3, and VT7/VP2 have been previously described ( , ). rVVs were propagated and titrated in African green monkey kidney epithelial BSC-1 cells (American Type Culture Collection) as described previously ( ).

    Techniques: Expressing, Infection, Metabolic Labelling, SDS Page, Electrophoresis, Autoradiography, Molecular Weight, Western Blot, Incubation

    N-linked glycosylation status of JUNV GPC. (A) Lysates of Vero cells infected at an MOI of 5 with either rRom (Romero) or rCan (Candid) were treated with PNGase F to remove N-linked oligosaccharides from protein backbone. The GPC expression profiles were analyzed by Western blotting using an anti-G2 antibody. (B) Activation of the UPR in HEK 293 cells expressing GPC of JUNV strains differing in in vivo attenuation status. Cells were transfected with equal amounts of expression plasmids for the GPC of Rom (Romero), Can (Candid), Can predecessor strains XJ13 and XJ44, and the GPC of Can and Rom bearing I427F (CanI427F) and F427I (RomF427I) amino acid changes in the G2 subunit, and A168T and T168A amino acid changes in the G1 subunit (C) , respectively. Cell lysates were harvested at 24 h post-transfection and divided into two equal aliquots that were either mock-treated (top panels) or treated with PNGase F (PNGase F treated). Expression of viral GPC and the UPR marker BiP was analyzed by Western blotting. 293, HEK 293 cells transfected with a GFP expressing construct that shares the same plasmid backbone with the expression constructs for JUNV GPC. Densitometry was calculated using AlphaEase TM in order to determine saturation of each band. Band saturation of the selected area is represented on a scale from 0 to 255. (C) GPC expression profiles in cells with or without the T168A substitution. Cells were transfected with equal amounts of each plasmid and allowed to incubate for 36 h. HEK 293 cell were transfected with a GFP plasmid containing the same backbone as the GPC expression plasmids. The cells were lysed and analyzed by Western blotting.

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Absence of an N-Linked Glycosylation Motif in the Glycoprotein of the Live-Attenuated Argentine Hemorrhagic Fever Vaccine, Candid #1, Results in Its Improper Processing, and Reduced Surface Expression

    doi: 10.3389/fcimb.2017.00020

    Figure Lengend Snippet: N-linked glycosylation status of JUNV GPC. (A) Lysates of Vero cells infected at an MOI of 5 with either rRom (Romero) or rCan (Candid) were treated with PNGase F to remove N-linked oligosaccharides from protein backbone. The GPC expression profiles were analyzed by Western blotting using an anti-G2 antibody. (B) Activation of the UPR in HEK 293 cells expressing GPC of JUNV strains differing in in vivo attenuation status. Cells were transfected with equal amounts of expression plasmids for the GPC of Rom (Romero), Can (Candid), Can predecessor strains XJ13 and XJ44, and the GPC of Can and Rom bearing I427F (CanI427F) and F427I (RomF427I) amino acid changes in the G2 subunit, and A168T and T168A amino acid changes in the G1 subunit (C) , respectively. Cell lysates were harvested at 24 h post-transfection and divided into two equal aliquots that were either mock-treated (top panels) or treated with PNGase F (PNGase F treated). Expression of viral GPC and the UPR marker BiP was analyzed by Western blotting. 293, HEK 293 cells transfected with a GFP expressing construct that shares the same plasmid backbone with the expression constructs for JUNV GPC. Densitometry was calculated using AlphaEase TM in order to determine saturation of each band. Band saturation of the selected area is represented on a scale from 0 to 255. (C) GPC expression profiles in cells with or without the T168A substitution. Cells were transfected with equal amounts of each plasmid and allowed to incubate for 36 h. HEK 293 cell were transfected with a GFP plasmid containing the same backbone as the GPC expression plasmids. The cells were lysed and analyzed by Western blotting.

    Article Snippet: Cells and viruses African green monkey kidney epithelial Vero, human embryonic kidney (HEK) 293, and baby hamster kidney (BHK-21) cells (American Tissue Culture Collection) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% (Vero and HEK 293) and 5% (BHK-21) fetal calf serum (HyClone) and an antibiotic-antimycotic solution (Gibco).

    Techniques: Gel Permeation Chromatography, Infection, Expressing, Western Blot, Activation Assay, In Vivo, Transfection, Marker, Construct, Plasmid Preparation