FIG. 5.

PML-dependent upregulation of TopBP1 by IR. (A and D) Effects of inhibition of PML expression by siRNA on IR-induced expression of TopBP1. U2OS cells were transfected with siRNA as described in Materials and Methods and cultured for 48 h. Cells were then exposed to 10 Gy of IR and harvested after an additional 8 h. Total proteins were isolated for Western blot analysis (A) or fixed on slides for immunofluorescence staining (D). (B and F) TopBP1 fails to form IR-induced foci in the PML-deficient NB4 cells and express low levels of TopBP1. NB4 cells with or without treatment with 1 μM ATRA for 72 h were irradiated with 10 Gy of IR. (F) Cells were then collected and immobilized on slides by cytospin, and immunofluorescence staining was performed with TopBP1 monoclonal and PML polyclonal antibodies. Cell nuclei were visualized by staining with DAPI. (B) For Western blot analysis, NB4 cells were treated with ATRA and IR as described above, and total proteins were isolated 12 h after IR. Each lane was loaded with 200 μg of protein, except for the SiHa cells, in which only 100 μg of protein per lane was loaded. (C) IFN fails to induce TopBP1 expression in PML^−/− MEFs. PML^−/− MEFs and normal MEFs were treated with 2,000 U of IFN-α/ml for 24 h. Total proteins were isolated, and Western blot analysis was performed with PML and TopBP1 antibodies. (E) IFN induced colocalization of PML and TopBP1. SiHa cells treated with 2,000 U of IFN-α/ml or untreated were fixed on slides. Double color immunofluorescence staining was performed with polyclonal PML antibody and monoclonal TopBP1 antibody. (G) Adenovirus-mediated reexpression of PML in PML^−/− MEFs induced TopBp1 expression. PML^−/− MEFs were infected at a multiplicity of infection of 25 with Ad-PML as described previously (12, 21). Double-color immunofluorescence staining was performed with the PML polyclonal antibody and TopBP1 monoclonal antibody.