Fig 1: Qualitative and quantitative analyses of HCMV AD169-GFP ?UL53 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 ?UL53. (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 ?UL53 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.