Journal: Cells

Article Title: ‘Shared-Hook’ and ‘Changed-Hook’ Binding Activities of Herpesviral Core Nuclear Egress Complexes Identified by Random Mutagenesis

doi: 10.3390/cells11244030

Figure Lengend Snippet: Qualitative and quantitative analyses of HCMV AD169-GFP ΔUL53 virus reconstitution on the different recombinant HFF cell populations. HFFs that express pUL53, pUL53-Flag or pUL53::sHook1-Flag were transfected with the infectious bacterial artificial chromosome of HCMV ΔUL53. ( A ) Images of the GFP signals and the respective brightfield illuminations were taken at indicated time points; scale bar in panel A, picture 1 marks 500 μm. ( B ) Quantitative analysis of fluorescence-positive cells was achieved with automated counting by the CellReporterXpress ® software (Molecular Devices LLC, San Jose, CA, USA). Counting parameters for positive nuclei were set to an intensity of at least 100, a minimal width of 7 and a maximal width of 30. Measurements were performed in biological sextuplicates per cell population of 5.05% area of the well and mean values ± SD are given. ( C ) qPCR-based assay for the determination of viral genome equivalents referring to the respective recombinant HFF populations. Viral supernatants were harvested at day 24 p.t. and subjected to IE1-specific qPCR. Calculations were performed in biological sextuplicates per cell population; mean values ± SD are shown. ( B , C ) Statistical analysis was performed using an ordinary two-way ANOVA and post-hoc Tukey correction; ****, p < 0.0001; **, p < 0.01; *, p < 0.1; ns, not significant; no significant differences were noted for values between 1–20 d p.t. ( B ). ( D ) Wb-based expression analysis of the conditionally expressing HFF populations. Recombinant protein expression in the three HFF populations of HFF-UL53, HFF-UL53-Flag and HFF-UL53::sHook1-Flag cells was either uninduced (-) or induced (500 ng/mL Dox). Cells were either infected with HCMV ΔUL53 or remained mock-infected. At 24 h p.t., cells were harvested and lysed. Total lysate samples were subjected to standard Wb analysis using tag-specific or protein-specific monoclonal antibodies as indicated.

Article Snippet: For measurement of HCMV AD169-GFP ΔUL53-positive cells, signals were counted with the ImageXpress ® Pico device (Molecular Devices LLC, San Jose, CA, USA) using CellReporterXpress ® software (version 2.9.3.1183, Molecular Devices LLC) by the system-integrated cell count assay.

Techniques: Virus, Recombinant, Transfection, Fluorescence, Software, Expressing, Infection, Bioprocessing

Journal: Cells

Article Title: ‘Shared-Hook’ and ‘Changed-Hook’ Binding Activities of Herpesviral Core Nuclear Egress Complexes Identified by Random Mutagenesis

doi: 10.3390/cells11244030

Figure Lengend Snippet: Viral replication kinetics of HCMV ΔUL53 and its revertant (HCMV Rev) determined by quantitation of GFP-positive cells and HCMV-specific qPCR on the different recombinant HFF populations. 80,000 inducibly expressing HFFs in 24-well plates were infected with HCMV ΔUL53 or HCMV Rev at a viral dose of 5 × 10 6 genome copies. pUL53, pUL53-Flag or pUL53::sHook1-Flag protein expression was either Dox-induced (+Dox) or remained non-induced (−Dox). ( A ) The number of HCMV-infected cells was measured by detection of GFP signal-positive cells at indicated time points with the CellReporterXpress ® software using the ImageXpress ® Pico device. Values represent 25.04 % of the area of a well and are given as a mean value ± SD of two independently infected wells. ( B ) Viral supernatants were harvested at indicated time points and viral genome equivalents were determined by qPCR. Each value represents the mean ± SD of two independent biological replicates, each measured twice. ( A , B ) Statistical analysis was performed using an ordinary two-way ANOVA and post-hoc Sidak correction; ****, p < 0.0001; ***, p < 0.001; *, p < 0.1; ns, not significant; no significant differences were noted for values between 1–13 d p.i. ( A ) or 1–11 d p.i. ( B ), respectively.

Article Snippet: For measurement of HCMV AD169-GFP ΔUL53-positive cells, signals were counted with the ImageXpress ® Pico device (Molecular Devices LLC, San Jose, CA, USA) using CellReporterXpress ® software (version 2.9.3.1183, Molecular Devices LLC) by the system-integrated cell count assay.

Techniques: Quantitation Assay, Recombinant, Expressing, Infection, Software