Fig. S1.

Setup and results of the SILAC experiment. (A) Experimental procedure for SILAC-based strategy. HeLa cells were cultured in medium containing light, medium, or heavy isotopes for 30 d. Cells were then transduced for 2 h with empty VLP, VLP containing Vpr S79A or Wt Vpr. Two independent biological replicates were performed named Silac (S1) and Switch (S2), in which isotopic conditions were inverted. At 12 h, treated cells were mixed in 1:1:1 ratio, separated into cytoplasmic and nuclear fractions, and subjected to proteomics experiments. (B) Vpr expression in HeLa cells treated with the VLP used for SILAC. HA-Vpr expression level was checked by Western blot. (C) WT Vpr-containing VLP used for SILAC trigger a G2 arrest. An aliquot of cells used for SILAC was fixed and then stained with propidium iodide, and DNA content was monitored by flow cytometry. The histogram displays the ratio between cells in G2/M and cells in G1 phases. (D) Identification of a new Vpr target by SILAC. The values were normalized taking as 100% the mock condition treated with empty VLPs. Two experiments were performed: Silac (S1) and Switch (S2), the values displayed for down- and up-regulated proteins by HIV-1 Vpr are the means of S1 and S2 ± SD. Only proteins with at least 20% variation are shown. *Group of peptides shared by several proteins. (E) Representation of significantly down- and up-regulated proteins by HIV-1 VprS79A compared with the mock condition (empty VLP). The values displayed for down- and up-regulated proteins by HIV-1 Vpr are the means of S1 and S2 ± SD. Only proteins with at least 20% variation are shown.