Vpr-mediated up-regulation of ULBP2 requires the recruitment of the DDB1-CUL4A (VprBP) E3 ligase complex and activation of the DNA damage/stress checkpoint arrest initiated by ATR. (A) CEM.NKR T cells were transduced with lentiviral vectors expressing GFP alone (WPI) or coexpressing GFP and VprWT (WPI-VprWT) or Vpr mutants (WPI-VprR80A or WPI-VprQ65R), as indicated. At 48 hours after transduction, GFP-expressing cells were monitored for ULBP-2 cell-surface expression by flow cytometry using specific mAbs directed against ULBP-2 and appropriate fluorochrome-conjugated secondary reagents (top panel). Expression of VprWT and Vpr mutants (VprR80A and VprQ65R) was evaluated by intracellular staining and flow cytometry using anti-Vpr mAbs and appropriate fluorochrome-conjugated secondary reagents (bottom panel). (B-C) CEM.NKR T cells were transduced with lentiviral vectors WPI or WPI-VprWT or treated with APC (4μM), in the presence or absence of caffeine (2.5mM) (B) and in the presence of DMSO or KU55933 (10μM) (C) as indicated. Cell-surface expression of ULBP-2 was monitored on GFP-expressing cells 48 hours after transduction or on the total cell population after a 24-hour treatment with APC. MFI values were calculated by subtracting the corresponding isotype control values (dashed line). Results shown are representative of the data obtained from at least 2 independent experiments. (D) HeLa cells were irradiated with γ rays (10 Gy from a Cs¹³⁷ source) in the presence of DMSO or KU55933 (10μM). Cells were then lysed and sonicated 1 hour after irradiation, and phosphorylation of Chk2 and H2AX was monitored by Western blotting using specific Abs.