Depletion of either SAP145 or SAP49 induces formation of γ-H2AX and BRCA1 foci. (A) HeLa cells were transfected with vector (GFP), GFP-Vpr, or GFP-Vpr-ΔC. At 48 h after transfection, the cells were stained with γ-H2AX- or BRCA1-specific antibodies (red). (B) Cells were transfected with either luciferase (control), SAP145, or SAP49 siRNAs. At 48 h after transfection, cells were stained with γ-H2AX- or BRCA1-specific antibodies (red). (C) γ-H2AX- and BRCA1-positive and γ-H2AX- and BRCA1-negative cells were visually counted. As a positive control, cells were treated with 10 mM hydroxyurea for 2 h. Cells were transfected with several vectors or siRNAs as shown in panels A and B. The results were averaged over three independent experiments (>200 cells each); bars indicate the standard deviations. (D) Immunoblot analysis with acid-soluble extracts (middle and bottom) or whole-cell extract (top) from HeLa cells transfected with either GFP (control), GFP-Vpr, or GFP-Vpr-ΔC plasmids or HeLa cells stably expressing HA-tagged SAP49, transfected with either luciferase (control), SAP145, or SAP49 siRNAs for 0, 24, 48, or 72 h. Blots were probed with anti-γ-H2AX (top), anti-GFP (Vpr), anti-SAP145, or anti-HA (SAP49) antibodies. Proteins were stained with Coomassie blue (bottom).