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. 2014 Sep 25;15(11):1163–1174. doi: 10.15252/embr.201439434

Figure 3. SET8 can localize to and promote 53BP1’s accumulation at DSBs independently of PCNA.

Figure 3

A Schematic of SET8 protein and the truncation forms.

B The N-terminus of SET8 is sufficient for DSB localization. U2OS cells were transfected with the indicated constructs.

C Immunoblots showing knockdown efficiency in whole-cell extracts derived from U2OS-ER-AsiSI cells transfected with non-targeting or PCNA-targeting siRNAs.

D–F ChIP-qPCR experiments were performed in U2OS-ER-AsiSI cell line transfected with the indicated siRNAs using anti-γH2AX, anti-SET8 and anti-H4K20me1 antibodies. P ≫ 0.05 (paired t-test) for difference in γH2AX levels at DSBs in siCON and siPCNA treated with OHT. P = 0.002 (paired t-test) for reduction in SET8 levels in siPCNA cells compared to siCON cells. P ≫ 0.05 (paired t-test) for reduction in H4K20me1 in siPCNA cells compared to siCON cells. Mean proportional reduction in SET8 levels at DSBs in siPCNA cells treated with OHT compared to siCON cells treated with OHT is 0.61 with a standard deviation of 0.26.

G 53BP1 IRIFs are unaffected after PCNA depletion. Immunostaining of U2OS cells transfected with non-targeting or PCNA-targeting siRNAs. Cells were allowed to recover for 1 h after irradiation (10 Gy) prior to staining.

H, I Abrogation of 53BP1 IRIFs by depletion of endogenous SET8 can be rescued with a siRNA-resistant form of SET8 containing a PCNA interacting mutation. Immunofluorescence of U2OS cells treated with non-targeting or SET8-targetting siRNAs co-transfected with plasmids encoding GFP or siRNA-resistant SET8 with PCNA binding mutant.