Fig 2.

Triple depletion of hDaxx, PML, and Sp100 in HepaRG cells. HepaRG cells were transduced with lentiviruses expressing shRNAs against hDaxx (shD); hDaxx and PML (shDP); or hDaxx, PML, and Sp100 (shDPS) or a scrambled shRNA with no cellular target (shneg). (A) Immunoblot analysis of extracts from the generated cell lines demonstrating depletion of the respective proteins, compared to parental untransduced cells (HepaRG). Antibodies used for detection were anti-hDaxx serum D7810, anti-PML serum A301-167A, and anti-Sp100 serum SpGH. The loading control was antiactin serum A5060. (B) Verification of depletion by immunofluorescence using the same cell lines. Cells were simultaneously stained with rabbit anti-hDaxx 07-471 antibody, mouse anti-PML MAb 5E10, and rat anti-Sp100 r26 antibody. Secondary antibodies were Alexa Fluor 488 goat anti-rabbit, Alexa Fluor 633 donkey anti-mouse, and goat anti-rat Cy3 IgG antibodies. Bar, 10 μm.