Fig. 6.

DDB2 functions downstream of EZH2 in NER and in mediating cisplatin resistance in SCLC. a and b EZH2 knockdown impairs DDB2 recruitment to CPD lesions. HeLa cells were transfected with siRNAs targeting EZH2 or a NT control. The following day, both groups were transfected with FLAG-DDB2. Cells were UV-irradiated at 100J/m² through a micropore membrane, fixed, and stained for CPD lesions and FLAG-DDB2. Relative percent of CPD stained lesions that were positive for DDB2 staining, normalized to NT, was quantified (a). Representative images are shown (b). c and d FLAG-DDB2 rescues impaired resolution of CPD lesions that occur through EZH2 knockdown. H128 cells were transfected with siRNAs targeting EZH2 or a NT control. The following day, both groups were transfected with FLAG-DDB2. Cells were UV irradiated, and harvested immediately (0h) or after 12h of repair time. Slot blot analysis was performed. c Quantitation of percent of CPD lesions remaining over time as normalized to 0 hrs of repair time. d Representative slot blot. SYBR Gold signal indicates total DNA loaded. e FLAG-DDB2 alleviates the sensitization of SCLC to cisplatin upon EZH2 knockdown. H128 cells were transfected with siRNAs corresponding to EZH2, ERCC1 or a NT control. The following day, groups were transfected with FLAG-DDB2 or mock control. 48 hours post overexpression, cell viability analysis was performed. f Western blot of FLAG-DDB2 expression achieved in H128 corresponding to panels c-e. For a-b, quantification was achieved through 3 independent experiments counting at least 50 independent CPD lesion events per group (n=50) and representative images are shown. c-d, representative blots from 3 independent experiments (n=3) is shown). *** indicates p<0.001.