Fig. 6
SUMO2-PCNA suppresses transcription-induced DSBs. a Western blot analysis of PCNA and FLAG-PCNA in whole cell extracts (WCE) using an antibody against PCNA. Tubulin was used as a loading control. b Quantification of γH2AX-positive cells overexpressing the indicated FLAG-PCNA complexes with or without DRB treatment (100 µM). Each value in the graph represents the average value ± standard deviation (n > 200) per one representative experiment. Only cells with 5 or more γH2AX foci were counted as positive cells. The result was reproduced in two independent assays. c Representative fluorescence images of γH2AX foci (green) in HEK293T cells overexpressing the indicated FLAG-PCNA proteins with or without DRB treatment. Nuclei were counterstained with DAPI. Scale bar, 10 µm. d The average tail moments (horizontal lines) measured by the neutral comet assay using HEK293T cells overexpressing the indicated FLAG-PCNA constructs with or without DRB treatment. Each dot represents the tail moment of a single cell. At least 150 cells were analyzed per one representative experiment. The result was reproduced in three independent comet assays. e Western blot analysis of immunopurified γH2AX from formaldehyde-treated HEK293T cells for ChIP analysis. f ChIP analysis of the samples from e for γH2AX occupancy at FRA7K using IMMP2L3b primers. The result was reproduced in two independent assays. g ChIP analysis of γH2AX occupancy at indicated loci in HEK293T cells overexpressing FLAG-PCNA (KR) and treated with or without DRB. The result was reproduced in three independent assays. h Representative analysis of R-loop frequency at IMMP2L3b (left) and ACTB exon 3 (EX3; right) sites in HEK293T cells overexpressing the indicated FLAG-PCNA constructs. The isolated nucleic acids were treated with or without RNase H prior to R-loop immunoprecipitation. The result was reproduced in two independent assays. For all the ChIP experiments described above, each value represents the average value±standard deviation calculated from triplicate qPCR reactions per one representative experiment. p values were calculated using t-test analysis for statistically significant differences. p≤0.05 are indicated with an asterisk (*)
