Fig 6. Inhibition of Cdk2 abrogates T592 phosphorylation.

a) HFFs were pretreated for 5 h with the Cdk1 inhibitor CGP74514A (CGP), and then not infected or infected with AD169, in the presence of CGP. After removing virus inoculum, the inhibitor was newly added and kept until cell harvesting, at 3 dpi. Total or pT592-SAMHD1 levels were analyzed by immunoblotting, together with IE1/IE2 and p85 expression. A representative experiment out of five is shown. b) The relative amount of pT592-SAMHD1, normalized to that of p85, was determined by densitometric analysis and is relative to that of n.i. cells, which was arbitrarily set as 1. Data are expressed as mean ± SE of four independent experiments performed at 1 and 3 dpi. c) Effect of CGP on infectious virus production as measured by standard plaque assays on cell culture supernatants at 3 dpi. Results derive from seven experiments and are expressed as mean ± SE. d) HFFs were pretreated as in panel a) with the Cdk inhibitors RO-3306 (RO), KO-3861 (KO) or BMS-265246 (BMS) at the indicated concentrations (μM), and then not infected or infected with AD169, in the presence of the inhibitors. After removing virus inoculum, the drugs were newly added and kept until cell harvesting, at 1 dpi. Immunoblotting was performed as above. One representative experiment out of two is shown. e) HFFs were transfected with Cdk1 or Cdk2 siRNA or non-targeting siRNA (ctrl). The day after, cells were not infected or infected with AD169 at an MOI of 1 and harvested at 1 dpi. Immunoblotting was performed as above. One representative experiment out of two is shown. NT, not treated cells; DMSO, cells treated with the vehicle DMSO; *, unspecific band; black arrow, pT592-SAMHD1 specific band. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; *****, p< 0.00001.