Construction of a single lentivirus vector for simultaneous depletion of hDaxx, PML, and Sp100. Shown is a diagrammatic representation of the triple-shRNA vector construction strategy. For generation of a lentivirus for simultaneous depletion of hDaxx and PML (pLKO-shDP), the mouse RNA polymerase III U6 promoter (mU6) was amplified using PCR primers encoding an shRNA against hDaxx (shDaxx) and inserted into a lentiviral vector encoding an shRNA against PML (pLKO-shPML) under the control of the human U6 promoter (hU6). For construction of a lentivirus for simultaneous depletion of hDaxx, PML, and Sp100 (pLKO-shDPS), the RNA polymerase III 7SK promoter (7SKpro) was amplified from human genomic DNA and isolated from a subsequent PCR using primers encoding an shRNA against Sp100 (shSp100). All lentivirus vectors used in this study express puromycin-N-acetyltransferase (Puro-N-ac).