Fig 2: Depletion of CRL4DDB2 components DDB2, DDB1 and CUL4A decrease H3K56Ac levels(A) DDB2, H3K56Ac, H3K9Ac, H3K27Ac, H3K27Ac and H3K14 levels were examined in LFS-041B cell line and its derivative cell line with re-established DDB2 expression. (B) Cytoplasmic and chromatin fractions of LFS-041B and its derivative were obtained through a cellular protein fractionation protocol. Samples containing identical protein amounts from each fraction were examined by Western blotting for DDB2, H3K56Ac and other acetylated histone H3. Actin and Lamin B blots serve as the loading controls. (C) NHFs were transiently transfected with control (Ctrl) siRNA or siRNA targeting DDB2, DDB1 or CUL4A. Levels of DDB2, DDB1 and CUL4A as well as H3K56Ac were detected in whole cell Laemmli extracts by Western blotting.

Fig 3: Depletion of CUL4A decreases recruitment of CAF-1 to damage sites(A) HeLa cells were transfected with control (Ctrl) siRNA and CUL4A siRNA. CAF-1 p150 and γH2AX were visualized by immunofluorescence in transfected cells exposed to micropore UVR. Arrows point to typical immunofluorescent focus of CAF-1 p150 or γH2AX. Calibration bar is 10 μm. (B) The quantitative data of CAF-1 p150 and γH2AX foci counts from multiple experiments. (C) the CAF-1 p60 and γH2AX were visualized by immunofluorescence. (D) The quantitative data of CAF-1 p60 and γH2AX foci counts from multiple experiments. (E) CAF-1 p150 and γH2AX were visualized by immunofluorescence in siRNA-transfected cells after phleomycin treatment. Dashed circles indicate the immunofluorescence decorated cellular area. (F) Western blotting analysis of γH2AX levels in control and CUL4A siRNA-transfected HeLa cells. Actin blots serve as the loading controls.

Fig 4: Posttranslational modification of the APC/C by SUMOylation. a Alignment of the Cullin domains present in the human Cullin proteins and the Cullin-like domain of APC2: The NEDDylated lysine residue within the Cullin proteins is highlighted in red and is absent in APC2. b HeLa cells were treated with DMSO as a negative control (-) or 1 µM MLN4924 (+), an inhibitor of the NEDD8-activating enzyme, for 16 h. Lysates were analyzed by immunoblotting with anti-APC2 and anti-CUL4A antibodies. Ponceau S staining was performed to monitor equal loading. c HeLa cells and HeLa cells expressing His-tagged SUMO2 (His-SUMO2) were subjected to a His-pulldown (His-PD). Input and pulldown samples were analyzed by immunoblotting making use of anti-APC4 and anti-SUMO2/3 antibodies. Blots were stained with Ponceau S to monitor equal loading. At least three independent experiments were performed and representative results are shown

Fig 5: Interaction between DDB2 with PCNA is in part responsible for maintaining H3K56Ac level(A) Immunoprecipitation experiments were performed using soluble chromatin fractions made from parental HeLa and stable HeLa-DDB2 cells. The immunoprecipitates were analyzed by Western blotting for the presence of FLAG (DDB2), DDB1, PCNA, CUL4A and CAF-1 p60 and p150. (B) Alignment of PIP box and nearby sequences. Green letters highlight conserved amino acids; blue letters indicate additional conserved amino acids of PIP degron; red letters indicate amino acids in mutated PIP box of DDB2. (C) Mutation in DDB2 PIP box did not affect XPC ubiquitination. XPC protein and its modified species were examined in control and DDB2 vector-transfected and UV-irradiated LFS-41B cells. (D) H3K56Ac and actin levels were examined in control and DDB2 vector-transfected LFS-41B cells and quantitated using ImageJ software. The relative H3K56Ac level is normalized against the actin control.