Figure 1: Phosphorylated HBO1 is detected in ultraviolet-induced DNA damage sites.

(a) Representative images of immunostaining for CPD and HBO1 or HBO1 pS50/53. HBO1 was phosphorylated at sites of local ultraviolet irradiation. shCtl, shDDB2 and shXPC cells were irradiated with 12 J m⁻² ultraviolet through a membrane with 5-μm pores. Cells were cultured for 30 min. Anti-HBO1 and anti-HBO1 pS50/53 antibodies were used for co-staining with anti-CPD antibody. The definition of relative fluorescence is shown in Supplementary Fig. 1b. The relative pS50/53/CPD intensities of shDDB2 and shXPC are shown as % of shCtl cells. Scale bars, 5 μm. n=30 cells from three independent experiments; error bars indicate means±s.d. **P<0.01 (Student’s t-test). (b) HBO1 pS50/53 is detected on the chromatin until 2 h after ultraviolet irradiation. HEK293 cells were mock-treated or irradiated with 50 J m⁻² ultraviolet. Cell lysates were fractionated into soluble and chromatin-enriched samples and then subjected to western blotting with the indicated antibodies. (c) Inhibition of ATR reduced accumulation of HBO1 pS50/53 at the damage sites. Cells were incubated with ATM and/or ATR inhibitors for 2 h before local ultraviolet irradiation at 50 J m⁻². After 30 min, cells were fixed and immunostained with anti-DDB2 and anti-HBO1 pS50/53 antibodies. The relative pS50/53/DDB2 intensities of ATM and/or ATR inhibitor-treated cells are shown as % of DMSO control cells. Scale bars, 5 μm. n=50 cells from three independent experiments; error bars indicate means±s.d. **P<0.01 (Student’s t-test). (d) Phosphorylation of HBO1 Ser50 and Ser53 in G1 phase is dependent on XPC. Cells were irradiated 50 J m⁻² ultraviolet and cultured for 30 min. Cells were fixed and immunostained with anti-pS50/53 and anti-γH2AX antibodies. DNA was stained with DAPI. DNA contents and fluorescence intensities of pS50/53 or γH2AX in cells were quantified by the IN Cell Analyzer. Cells in G1 or S-G2/M phase were defined by DNA content. The average pS50/53 or γH2AX intensity in shXPC cells in G1 and S-G2/M phase is shown as % of shCtl cells. Cells (n=1,500) from three independent experiments; error bars indicate means±s.d. **P<0.01 (Student’s t-test).