Fig. 5.

EZH2 promotes the stability of DDB2 independent of its catalytic activity and PRC2. a-d EZH2 promotes the stability of DDB2. H128 cells were transfected with siRNAs targeting EZH2 or a NT control. 72 hours after transfection, cells were pre-treated with cycloheximide (CHX) for 2 hours, and UV irradiated at 30 J/m² (a), treated with 15 μM cisplatin (b), or left untreated (−). Cells were harvested after treatment as indicated. Whole cell lysates were run on SDS-PAGE and immunobloted with the indicated antibodies. c EZH2 knockdown does not alter mRNA levels of DDB2. H128 cells were transfected with siRNAs targeting EZH2 or a NT control and RT-PCR analysis was performed to measure DDB2 and GAPDH mRNA levels. The relative ratio of DDB2 to GAPDH mRNA level is represented. Mean and standard deviation of three replicas is shown. d H128 cells were transfected with siRNAs targeting EZH2 or a NT control. 72 hours post transfection, cells were pre-treated with 5 μM MG132 for 4 hours, and CHX for 2 hours for all groups. Cells were then UV irradiated at 30 J/m² and harvested at the indicated timepoints prior to SDS-PAGE and western blot analysis with indicated antibodies. e-i EZH2 stabilization of DDB2 is independent of its PRC2 function. e HCT116 cells were transfected with GFP-EZH2 WT or GFP-EZH2 catalytic inactive mutant (H689A) plasmids. 24 hours post transfection, cells were pre-treated with CHX for 2 hours, and left untreated (−) or UV irradiated at 30 J/m², harvested after UV treatment as indicated, run on SDS PAGE, and probed with indicated antibodies. f H128 cells were knocked down with siRNAs targeting the EZH2 5’UTR (EZH2-3) or a NT control, and the following day, transfected with GFP-EZH2 WT or GFP-EZH2 catalytic inactive mutant (H689A) or mock transfected. 48 hours post-transfection, groups were pre-treated with CHX for 2 hours, harvested, run on SDS PAGE, and probed with the indicated antibodies. g HCT116 cells were pre-treated with 1 μM SAM-competitive EZH2 inhibitor EPZ-6438 or DMSO for 4 days. Cells were then pre-treated with CHX for 2 hours. Cells were left untreated (−) or UV irradiated at 30 J/m² and harvested after 3 hours recovery. Whole cell lysates were run on SDS-PAGE and immunobloted with the indicated antibodies. h HeLa cells were transfected with with FLAG-EZH2 WT or PRC2 mutants (FLAG-EZH2 T311E/A). 48 hours post transfection, cells were pre-treated with CHX for 2 hours, and harvested. Whole cell lysates were run on SDS-PAGE and immunobloted with the indicated antibodies. i HCT116 Cells were transfected with siRNAs targeting SUZ12 or a NT control. 72 hours after transfection, cells were pre-treated with CHX for 2 hours, and left untreated (−) or UV irradiated at 30 J/m², and harvested after UV treatment as indicated. Whole cell lysates were run on SDS-PAGE and immunobloted with the indicated antibodies. j His-Ubi HEK-293T cells were transfected with indicated siRNA and 72 hours post transfection, cells were pre-treated with CHX and MG132 where indicated. Ubiquitinated DDB2 was measured. (For a-b and d-j, representative blots from 3 independent experiments (n=3) is shown).