Figure 3: XPC accumulation is decreased in HBO1-depleted cells.

(a) Accumulation of XPC at damage sites was suppressed in shHBO1 cells. 50 J m⁻² local ultraviolet-irradiated shCtl, shDDB2, shHBO1 and shXPC cells were incubated for 30 min and subjected to immunostaining with anti-DDB2 and anti-XPC antibodies. The relative XPC/DDB2 intensity of shHBO1 cell is shown as % of shCtl cells. Scale bars, 5 μm. Cells (n=50) from three independent experiments; error bars indicate means±s.d. *P<0.05 (Student’s t-test). (b–d) Live-cell imaging of XPC–EGFP accumulation at damage sites. Accumulation of XPC–EGFP was defective in shDDB2 cells and shHBO1 cells (b), shHBO1 cells stably expressing Myc-HBO1 EQ or SA (c), and ATM and ATR inhibitor-treated shCtl cells (d). XPC–EGFP was transiently transfected into shCtl, shDDB2 and shHBO1 cells and cells were irradiated with 405-nm laser in Hoechst 33342-containing medium. Quantification of accumulated XPC–EGFP was performed as described in Methods. Cells (n=30) from three independent experiments; error bars are the mean±s.e.m. *P<0.05, **P<0.01 (Student’s t-test). (e) ATM and ATR inhibitor treatment suppressed XPC accumulation at ultraviolet-induced DNA damage sites. Cells were local irradiated with ultraviolet 50 J m⁻² and cultured for 30 min. The relative XPC/DDB2 intensity of ATM and ATR inhibitor-treated cells is shown as % of DMSO control cells. Cells (n=50) from three independent experiments; error bars indicate means±s.d. **P<0.01 (Student’s t-test). (f,g) Relative XPC accumulation at damage sites at 20 and 60 min after 12 J m⁻² ultraviolet irradiation was quantified and shown as % of shCtl cells after 20 min ultraviolet. Cells (n=50) from three independent experiments; error bars indicate means±s.d. **P<0.01 (Student’s t-test).