Figure 6: Phosphorylated Ser50 and Ser53 of HBO1 at sites of ultraviolet-induced damage is involved in canonical GG-NER pathway.

(a) Fibroblasts derived from normal and XPE, XPC and XPA patients were irradiated with 12 J m⁻² local ultraviolet and cultured for 30 min. Cells were immunostained with anti-CPD and anti-HBO1 pS50/53 antibodies. The relative pS50/53/CPD intensities of 98BR, XP3KA and XP7HM cells are shown as % of levels in N25 cells. Scale bars, 5 μm. Cells (n=30) from three independent experiments; error bars indicate means±s.d. **P<0.01 (Student’s t-test). (b) Fibroblasts derived from normal and XPE, XPC and XPA patients were depleted for HBO1 by a shHBO1-expressing lentivirus. Cells were not irradiated (-ultraviolet) or irradiated with 12 J m⁻² ultraviolet and cultured for 0, 1, 8 and 24 h, and then fixed and immunostained with anti-CPD antibody. Relative intensities of CPD normalized against DAPI are shown as fold of ultraviolet 0 h cells. Cells (n=50) from three independent experiments; error bars indicate means±s.d. **P<0.01 (Student’s t-test). (c) Fibroblasts derived from normal and XPE, XPC, XPA and UVsSA patients were depleted for HBO1 by a shHBO1-expressing lentivirus. Cells were irradiated at the indicated ultraviolet dose, cultured for 2 weeks and then fixed with MeOH/acetic acid and stained with crystal violet solution. The number of colonies were counted and are represented as % of mock-irradiated cells. Three independent experiments were performed. Error bars indicate means±s.d.