Figure 3.

p97 partakes in the regulation of pS824-KAP1. (A) Time-dependent formation and degradation of pS824-KAP1 foci. The pS824-KAP1 foci were examined in irradiated MCF7, MCF7/shKAP1 and MCF7/shRNF4 cells (8 Gy). Cells were fixed at the indicated time points post-IR and pS824-KAP1 and γ-H2AX foci visualized by IF staining. (B) p97 is co-localized to where KAP1 and RNF4 interact. KAP1 (625-835)-VN173 and RNF4 (WT)-CC155 were co-transfected into U2OS cells. After 48-h, cells were pre-treated with MG132 (5 μM) for 4-h prior to IR exposure. Cells were fixed at 1-h following irradiation (4 Gy). Green: KAP1-RNF4 interaction; Red: p97. Scale bar: 5 µm. (C) Interaction of RNF4 with p97. HEK293 cells were co-transfected with SFB-RNF4 and p97-His-Myc followed by treating with Dox (3 μM) for the indicated time periods. Cell lysates were incubated with S-beads to pull down SFB-RNF4-associated complex followed by Western blot analysis. p97-His-Myc was detected by anti-Myc antibody. 5% input control was blotted with the indicated antibodies. (D) pS824-KAP1 signal was further enhanced in MCF7/shRNF4 cells treated with DBeQ or sip97. Cells were transfected with siRNA against p97 or treated with DBeQ (10 μM) in the presence of vehicle or Dox (3 μM). pS824-KAP1, total KAP1, pS1981-ATM, total ATM were assessed by blotting with the corresponding antibodies. β-actin served as loading control. (E) p97 inhibitor sensitizes MCF7 cells to IR. MCF7 cells were treated with DBeQ (200 nM) and increasing dose of IR. Colonies were fixed and stained with crystal violet at 12-days after treatment. Surviving fraction was calculated with a correction for the plating efficiency. Bars: mean ± SD; *: p < 0.02.