Fig 1: HCMV deubiquitinase inhibits anti-viral innate immunity to induce oncogenesis (a) Transcript of pan-interferon-α (P-IFNα) and (b) IFNβ was measured by qPCR upon WT-HCMV (H-WT) and ΔDUB-HCMV (HΔDUB) infection or poly(I:C) transfection to HFF cells, on 3 and on 6 dpi or dpt, as indicated. (c, d) Transcript of IFNβ was measured by qPCR upon H-WT infection to myd88 (c) or sting (d) knocked down HFFs. (e–g) Luciferase assay was done for IFNα4 and IFNβ promoters in HEK293 cells, as depicted. (h, i) Transcript level of P-IFNα and IP10 was measured in HeLa cells transfected with UL48N and stimulated with CpG. (j, top) NDV transcript level was measured in IMR32 cells transiently transfected with Vec, UL48N and UL48NΔDUB expression plasmids as depicted. (j, bottom) Overexpression of UL48N and UL48NΔDUB was detected by using anti-FLAG antibody. (k) MTT assay was done with HCMV-infected and rIFN-β treated HFFs as depicted and significance of WT- or HΔDUB infection group was calculated compared with mock. (l, left-top) Wound-healing was observed in IMR32 cells transiently transfected with increasing amount of Vec, UL48N and UL48NΔDUB expression plasmids and treated with constant amount of rIFN-β (10 U), as depicted, (l, right) wound size was measured and (l, left bottom) expression of UL48N and UL48NΔDUB was analyzed using anti-FLAG antibody. (m, n) Transcript of IFNβ and MKi67 was measured by qPCR upon anti-IFNAR2 antibody treatment and WT-HCMV (H-WT) or ΔDUB-HCMV (HΔDUB) infection to HFF cells, on 3 dpi as indicated. (o) MTT assay was done with HFF cells upon anti-IFNAR2 antibody treatment (for 4 h) and WT-HCMV (H-WT) or ΔDUB-HCMV (HΔDUB) infection to HFF cells, on 3 dpi as indicated. Vec: Empty Vector, NΔDUB: UL48NΔDUB, p(I:C)-T: Polyinosinic:polycytidylic-Transfection, dpi: days post infection, dpt: days post transfection, hpt: hours post transfection, hps: hours post-stimulation, hpi: hours post infection, dptr: days post treatment, sh: short-hairpin, rIFN: recombinant interferon, IFNAR: Type I interferon α/β Receptor. Shown results are the representative of two (a–d, and h–o) or three (e–g) independent experiments. 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). See also Supplementary Figure S2a–S2e
Fig 2: HCMV infection induces oncogenic properties in non-transformed HFFs. (a) GFP expression was observed under the fluorescence microscope at 24 h post infection (hpi) with GFP-tagged WT-HCMV (H-WT) in HFFs. (b) Culture media color change (red to yellow) was visually observed on day 6 post infection (dpi), in HFFs infected with H-WT. (c) Cell viability (metabolic activity) of mock or H-WT-infected HFFs was quantified by MTT assay, on 6 dpi. (d) Transcript and (e) protein of MKi67 was quantified by qPCR and flow cytometer respectively, in mock, H-WT-infected HFFs, (f, left, right, bottom) flowcytometry was done for mock and H-WT-infected HFFs by staining them with propidium iodide (PI, shown in x axis) on 3 dpi, to detect cell cycle stages upon HCMV infection and (g) Anti-apoptotic gene (bcl2, birc3 and prkce) status was compared by qPCR in mock or H-WT infected HFFs on 6 dpi. Shown results are the representative of three (a–d, g) or two (e, f) independent experiments. (f) Statistical analysis was done with data of two independent experiments. 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)
Fig 3: 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)
Supplier Page from Novus Biologicals, a Bio-Techne Brand for Ki67/MKI67 Antibody [Allophycocyanin]
Available conjugates: PE;PE/Cy7;Biotin;FITC;PE/Cy5.5;Allophycocyanin