Journal: Cell Biology and Toxicology

Article Title: JAG1 mediates apoptosis in herpes simplex keratitis by suppressing autophagy via ROS/JAG1/NOTCH1/pULK1 signaling pathway

doi: 10.1007/s10565-024-09968-0

Figure Lengend Snippet: Antibodies

Article Snippet: HSV-1 ICP0 (mouse) , WB , Santa Cruz, USA , sc-53070.

Techniques:

Journal: Cell Biology and Toxicology

Article Title: JAG1 mediates apoptosis in herpes simplex keratitis by suppressing autophagy via ROS/JAG1/NOTCH1/pULK1 signaling pathway

doi: 10.1007/s10565-024-09968-0

Figure Lengend Snippet: HSV-1 infection induces apoptosis in HCECs and mice corneal epithelium. a – f HCECs were infected with HSV-1 at different MOIs (MOI = 0.5, 1, and 2) for 24 h. a Representative western blot analysis of virus-related proteins ICP4, ICP0, HSV-1 gD, and apoptosis-related proteins BAX and Bcl-2. b Statistical analysis of a ( n = 3). c Representative immunofluorescence staining of HSV-1 gD (green) and DAPI (blue) in HCECs (bars = 100 µm). d Representative FACS assessed the rate of apoptotic cells. e Statistical analysis of d ( n = 5). f Representative TUNEL staining (red) and DAPI (blue) of HCECs (bars = 200 µm). g Representative images of anterior segment and fluorescein staining of mice corneas. h Representative H&E staining of mice corneas (bars = 100 µm). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001

Article Snippet: HSV-1 ICP0 (mouse) , WB , Santa Cruz, USA , sc-53070.

Techniques: Infection, Western Blot, Virus, Immunofluorescence, Staining, TUNEL Assay

Journal: Frontiers in Immunology

Article Title: US6 Gene Deletion in Herpes Simplex Virus Type 2 Enhances Dendritic Cell Function and T Cell Activation

doi: 10.3389/fimmu.2017.01523

Figure Lengend Snippet: ΔgD-2 HSV type 2 (HSV-2) is attenuated in dendritic cells (DCs). (A) DC viability, as determined by flow cytometry (gated on CD11c + , I-A b+ , Zombie + cells) at different time points after virus inoculation with an multiplicity of infection (MOI) of 1. UT, untreated. (B) Western blot analyses of structural (VP16), early (ICP5, ICP27), and late (gB) viral proteins post-inoculation of DCs. (C) Viral genome loads in inoculated DCs determined by qPCR for the UL30 gene. Data are means ± SEM of three independent experiments. Representative images are shown for western blots. Two-way analysis of variance and Tukey’s multiple comparison test were used for statistical analyses (** p < 0.01).

Article Snippet: Viral protein expression was analyzed by western blot using primary antibodies against anti-ICP5 (Santa Cruz), anti-VP16 (Santa Cruz), anti-ICP27 (Santa Cruz), and anti-β-actin (BioLegend).

Techniques: Flow Cytometry, Virus, Infection, Western Blot, Comparison

Journal: Frontiers in Immunology

Article Title: US6 Gene Deletion in Herpes Simplex Virus Type 2 Enhances Dendritic Cell Function and T Cell Activation

doi: 10.3389/fimmu.2017.01523

Figure Lengend Snippet: Dendritic cells (DCs) inoculated with ΔgD-2 or wild-type (WT) virus mainly release defective herpes simplex virus particles. (A) Four predominant viral particle phenotypes, indicated in the four panels, were observed in the extracellular space of virus-inoculated DCs (extracellular space data shown). Transmission electron microscopy. Extreme left: WT-like particles displaying an envelope, an electron-dense tegument, and an electron-dense capsid; middle left: envelope-only phenotype displaying an envelope-like structure, harboring spikes in the outer region, yet no apparent capsid or tegument; middle right: WT-like capsids, yet no envelope; extreme right: empty capsids with no envelope and no electron-dense content in capsids. Right panel: quantification of viral particle phenotypes observed. (B) Transmission electron microscopy (negative staining) of viral particles recovered from ultracentrifugated supernatant of virus-inoculated DCs. (C) Western blot analysis of the major capsid protein ICP5 in virus particles recovered from ultracentrifugated supernatants of DCs 18 h post-virus inoculation. Values indicate relative expression of ICP5. (D) Quantification of viral genome copies (determined by qPCR) in viral particles obtained from ultracentrifugated supernatants derived from virus-inoculated DCs with or without acyclovir treatment. One-way analysis of variance and Tukey’s multiple comparison test were used for statistical analyses (* p < 0.05).

Article Snippet: Viral protein expression was analyzed by western blot using primary antibodies against anti-ICP5 (Santa Cruz), anti-VP16 (Santa Cruz), anti-ICP27 (Santa Cruz), and anti-β-actin (BioLegend).

Techniques: Virus, Transmission Assay, Electron Microscopy, Negative Staining, Western Blot, Expressing, Derivative Assay, Comparison

Journal: bioRxiv

Article Title: Transcription factor recruitment by parallel G-quadruplexes to promote transcription: the case of herpes simplex virus-1 ICP4

doi: 10.1101/2020.07.14.202747

Figure Lengend Snippet: (A) CD spectra of the biotinylated oligonucleotides used in the cross-linking-pull-down assay, folded in potassium phosphate buffer (20 mM PB, 80 mM KCl). (B) Western Blot analysis of ICP4 binding to the indicated G4s (viral: un2L2, un2, LTR-III, LTR-II+III+IV; cellular: myc). Infected cell nuclear extracts (5 μg) were incubated with formaldehyde-activated G4-folded oligonucleotides; proteins bound to the G4s were eluted after high stringency washes. ICP4 was detected by the anti-ICP4 antibody and CTR is a control lane loaded with infected cell nuclear extracts (3.5 μg) not subjected to the pull-down procedure. MW: molecular weight (Marker VI, Applichem). Below the gel image, the 260/290 ratio for each biotinylated oligonucleotide is displayed. Quantification of ICP4 bound to the G4s is indicated in the bar chart (average of two independent experiments, SD is displayed). Band quantification was normalized for the protein amount loaded per well and to the CTR sample (5 μg in G4 lanes, 3.5 μg in CTR). (C) First derivative FRET-melting curves (–d F 525 /dT vs T) of F-un2-T G4 oligonucleotide treated with ICP4 or BSA at the indicated protein/DNA ratios, in 20 mM PB pH 7.4, 2 mM KCl. ( D ) CD spectra showing the unfolding (molar ellipticity reduction) of un2L2 G4 in the presence of increasing ufICP4 concentrations. The protein spectrum was subtracted from DNA-protein complex spectra.

Article Snippet: Briefly, 10 μg of anti ICP4 antibody (mouse monoclonal H943, Santa Cruz Biotechnology, Dallas, TX, USA) was coupled to Protein A/G Plus Agarose for 1 h and subsequently cross-linked for 30 minutes (following procedures described in section A and B of the protocol, Pierce® Cross-link Immunoprecipitation Kit Manual).

Techniques: Circular Dichroism, Pull Down Assay, Western Blot, Binding Assay, Infection, Incubation, Control, Molecular Weight, Marker

Journal: bioRxiv

Article Title: Transcription factor recruitment by parallel G-quadruplexes to promote transcription: the case of herpes simplex virus-1 ICP4

doi: 10.1101/2020.07.14.202747

Figure Lengend Snippet: (A) Representative images of immuno-DNA-FISH in U-2 OS cells infected with HSV-1, strain F, MOI of 3, fixed at 8 hpi. The green and red signals correspond to un2 G4 and anti-G4 Ab signals, respectively; the merged signals are in yellow. Controls are shown in Fig. S5; (B) Representative images of proximity ligation assay (PLA) in U-2 OS cells infected with HSV-1, strain F, MOI of 3, fixed at 8 hpi. The green dots (PLA signal) correspond to G4-ICP4 interaction (one or two dots per nucleus). The blue signal corresponds to TOTO3 nuclear staining. Controls are shown in Fig. S6.

Article Snippet: Briefly, 10 μg of anti ICP4 antibody (mouse monoclonal H943, Santa Cruz Biotechnology, Dallas, TX, USA) was coupled to Protein A/G Plus Agarose for 1 h and subsequently cross-linked for 30 minutes (following procedures described in section A and B of the protocol, Pierce® Cross-link Immunoprecipitation Kit Manual).

Techniques: Infection, Proximity Ligation Assay, Staining

Journal: bioRxiv

Article Title: Transcription factor recruitment by parallel G-quadruplexes to promote transcription: the case of herpes simplex virus-1 ICP4

doi: 10.1101/2020.07.14.202747

Figure Lengend Snippet: (A) Western blot analysis of ICP4 cross-linking pull-down towards the indicated four G4s embedded in the ICP4 promoter. IE-CS is the reported ICP4 consensus sequence ( ; ; ); SCR is a G-rich unfolded oligo; CTR is a control lane loaded with infected cell nuclear extracts not subjected to the pull-down procedure. The upper panel shows the unbound ICP4 fraction, the middle panel the washed out ICP4 fraction, the lower panel the ICP4 fraction bound to each oligo. The multiband appearance of ICP4 from HSV-1 infected cells in WB has been reported and reflects the presence of differently post-transcriptionally processed isoforms. (B) CD spectra of the indicated G4 sequences embedded in the ICP4 promoter folded in potassium phosphate buffer (20 mM PB, 2 mM KCl) and incubated in the absence/presence of uflICP4 (10x). ICP4 spectrum was subtracted from G4-ICP4 complex spectra.

Article Snippet: Briefly, 10 μg of anti ICP4 antibody (mouse monoclonal H943, Santa Cruz Biotechnology, Dallas, TX, USA) was coupled to Protein A/G Plus Agarose for 1 h and subsequently cross-linked for 30 minutes (following procedures described in section A and B of the protocol, Pierce® Cross-link Immunoprecipitation Kit Manual).

Techniques: Western Blot, Sequencing, Control, Infection, Circular Dichroism, Incubation

Journal: bioRxiv

Article Title: Transcription factor recruitment by parallel G-quadruplexes to promote transcription: the case of herpes simplex virus-1 ICP4

doi: 10.1101/2020.07.14.202747

Figure Lengend Snippet: (A) Western blot analysis of ICP4 expression in the presence of increasing amounts of ICP4 expression plasmid in transfected U-2 OS cells. ICP4 and TK promoters were cloned in the promoterless pGL-4.10 plasmid upstream of the luciferase gene and transfected into U-2 OS cells. β-actin was used as housekeeping control for cell amount (B) Luciferase reporter assay of ICP4 and TK promoter activity in the presence of increasing amounts of ICP4 protein from ICP4 expression plasmid.

Article Snippet: Briefly, 10 μg of anti ICP4 antibody (mouse monoclonal H943, Santa Cruz Biotechnology, Dallas, TX, USA) was coupled to Protein A/G Plus Agarose for 1 h and subsequently cross-linked for 30 minutes (following procedures described in section A and B of the protocol, Pierce® Cross-link Immunoprecipitation Kit Manual).

Techniques: Western Blot, Expressing, Plasmid Preparation, Transfection, Clone Assay, Luciferase, Control, Reporter Assay, Activity Assay

Journal: bioRxiv

Article Title: Transcription factor recruitment by parallel G-quadruplexes to promote transcription: the case of herpes simplex virus-1 ICP4

doi: 10.1101/2020.07.14.202747

Figure Lengend Snippet: (A) Luciferase reporter assay of ICP4 promoter and control TK promoter activity in the presence of increasing B19 concentrations. ICP4 and TK promoters were cloned in the promoterless pGL-4.10 plasmid upstream of the luciferase gene and transfected into U-2 OS cells. U-2 OS cell viability in the presence of increasing concentrations of B19 was also evaluated. (B) Luciferase reporter assay of ICP4 and TK promoter activity in the presence of ICP4 expression plasmid and in the presence/absence of B19 added 2 h after (AT) or before (BT) transfection of reporter plasmid. (C) Upper panel: representative confocal microscopy images of U-2 OS cells transfected with ICP4-EYFP plasmid that express EYFP under the ICP4 promoter, in the presence of increasing concentrations of B19 (0, 5, 10, 20 μM, as indicated). Lower panel: quantification expressed as corrected total cell fluorescence (CTCF) of cell images, calculated on 6-8 slides (50-100 nuclei), mean average levels from two independent experiments. (D) Western blot analysis (left) and quantification (right) of the viral ICP4 protein in the presence of increasing concentrations of B19 in U-2 OS cells infected with HSV-1 strain F (MOI 1). MW: molecular weight (Marker VI, Applichem). ICP4 bands were quantified with respect to alpha-tubulin (housekeeping) levels in each lane. Mean average values of two independent experiments.

Article Snippet: Briefly, 10 μg of anti ICP4 antibody (mouse monoclonal H943, Santa Cruz Biotechnology, Dallas, TX, USA) was coupled to Protein A/G Plus Agarose for 1 h and subsequently cross-linked for 30 minutes (following procedures described in section A and B of the protocol, Pierce® Cross-link Immunoprecipitation Kit Manual).

Techniques: Luciferase, Reporter Assay, Control, Activity Assay, Clone Assay, Plasmid Preparation, Transfection, Expressing, Confocal Microscopy, Fluorescence, Western Blot, Infection, Molecular Weight, Marker