Fig. 6.

Reduced binding of XPC to CPD sites upon ASH1L depletion. a Scheme illustrating the experimental strategy to monitor the dynamics of XPC protein in the chromatin of living cells. At the time of analysis, the cells display small UV spots (generated with 3-µm pore filters) containing only CPDs and large UV spots (generated with 5-µm pore filters) containing a mixture of both 6–4PPs and CPDs. b ASH1L depletion compromises the ability of XPC protein to associate with the CPDs in small spots. Transfections with siRNA occurred 48 h before UV radiation of U2OS cells through 3-µm filter pores. Immunofluorescence was assessed 15 min after the second UV treatment through 5-µm filter pores. Scale bars = 3 µm and 5 µm. Quantifications show the amount of cells containing small XPC spots (n = 4 with 200 cells in each experiment); error bars show s.e.m. (two-tailed t-test). c Binding of XPC, XPD and DDB2 to chromatin upon UV radiation. HeLa cells were transfected with siASH1L or siNC, exposed to UV light (10 J m^–2) 48 h later, and analyzed by immunoblotting at different times. Chromatin was salt-extracted to release free proteins. GAPDH and H3 were the loading controls for free proteins and chromatin, respectively. d Level of XPC, XPD and DDB2 in chromatin, relative to the respective free proteins, normalized to H3 and GAPDH (n = 4 independent experiments, two-tailed t-test). These quantifications include both unmodified and ubiquitinated XPC protein and, by showing relative levels, take into account the overall higher XPC content of ASH1L-depleted cells