Figure 3
HCMV deubiquitinase induces cancer hallmarks: (a, b) HFFs were infected with equal MOI of GFP-tagged WT-HCMV (H-WT) and ΔDUB-HCMV (HΔDUB) and analyzed for apoptosis-associated genes on 6 dpi, (a) transcript level of anti-apoptotic genes bcl2, birc3, prkce, survivin and xiap as well as (b) transcript level of pro-apoptotic genes trail, Rb, p53, fadd and tnfα was measured by qPCR in mock, H-WT and HΔDUB-infected HFFs. (c) Endogenous protein level of BCL2, BIRC3, Survivin, TP53, Caspase-3 and p21 was analyzed in HFFs upon 8 dpi with H-WT and HΔDUB (d) HCMV-infected HFF cells were treated with 30μM etoposide on 3 dpi and analyzed for apoptosis rescue 24 h later by quantifying Annexin V level through flow cytometer. (e, left, right) IMR32 cells stably expressing Vec, UL48N and UL48NΔDUB were glucose starved for 2 h and treated with FITC-labeled Glucose for 10 min and then subjected to flow cytometer to observe the glucose uptake (f, top) Wound was created in IMR32 cells transiently transfected with Vec, UL48N and UL48NΔDUB expression plasmids as depicted and healing was observed for 48 h, (f, bottom) wound size was measured. (g, left) 5 × 105 IMR32 cells transiently transfected with Vec, UL48N and UL48NΔDUB expression plasmids were resuspended in serum deprived growth media and inoculated onto the matrigel layer containing insert, which was further kept in contact with serum containing growth media. Tissue invasion of these IMR32 cells was analyzed through matrigel invasion. (g, right) Matrigel invaded cells were counted and graph was plotted. Blue dots represent the invaded cells. Vec: Empty Vector, dpi: days post infection, NΔDUB: UL48NΔDUB, Etp: Etoposide, FITC-A: FITC-Area. Shown results are the representative of three (a, b, d, e, f, g) or single (c) 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 S1a–S1l
