Figure 2

HCMV deubiquitinase is responsible for induction of oncogenic properties. (a) HFFs were infected with equal MOI of GFP-tagged H-WT and HΔDUB; equal amount of GFP expression was microscopically observed at 24 h post infection (hpi) with GFP-tagged H-WT and HΔDUB in HFFs. Quantification of GFP fluorescence (HCMV infection) was done with flow cytometer; histogram shows the percentage of HCMV infection at 24 hpi, measured by flowcytometry and bar- graph shows the GFP-Mean Fluorescent Intensity (MFI) of the respective histograms. (b) Culture media color change (red to yellow) was visually observed on 6 dpi, in HFFs infected with H-WT. (c) Cell viability (metabolic activity) was quantified and compared among mock, H-WT and HΔDUB-infected HFFs, by MTT assay. (d) Transcript of proliferation marker gene MKi67 was quantified by qPCR in mock, H-WT and HΔDUB-infected HFFs. (e, left) MKi67 protein analysis was done in mock, H-WT and HDUB-infected HFFs by flowcytometry (e, right) MFI was calculated for respective MKi67 histograms. (f) Proliferation rate of Mock, H-WT and HΔDUB-HCMV-infected HFFs was analyzed by seeding them into equal number, harvesting them on 3 dpi and counting them to compare the total number of cells. (g, left, right, bottom) Cell Cycle stages of mock, H-WT and HΔDUB-infected HFFs were analyzed by flowcytometry by staining these HFFs with propidium iodide (PI) (shown in x axis) on 3 dpi. (h, left, right) The cell proliferation rate of IMR32 cells, stably expressing Vec, UL48N and UL48NΔDUB, was compared by initially seeding them into a density of 0.1 × 10⁶ cells, followed by counting and re-seeding them on every alternate day for 6 days. (i, left, right) Cell Cycle stages IMR32 stably expressing vector UL48N or NΔDUB were analyzed by flowcytometry by staining the cells with PI. dpi: days post infection, Vec: Empty Vector, NΔDUB: UL48NΔDUB. Shown results are the representative of three (a–d, f, h, i) or two (e, g) independent experiments. (g, h) Statistical analysis was done with data of two (g) and three (h) independent experiments and the difference was calculated between mock versus WT-HCMV infection or mock versus HΔDUB-HCMV infection (g) or between Vec versus UL48N or Vec versus NΔDUB (h). Differences were considered statistically significant with a *P-value<0.05, **P-value<0.01 and ***P-value<0.001, ns, non-significant difference (P-value>0.05)