Figure 3.

p97 inhibition results in a change from error-free homologous recombination (HR) to mutagenic repair by RAD52

(A and B) Representative images (A) and quantification (B) of the number of RAD51 foci of EdU-positive T24 cells at 4 h after 2 Gy IR and CB-5083 treatment. Scale bars, 5 μm; n = 3.

(C and D) Representative images (C) and quantification (D) of the number of RAD52 foci of EdU-positive T24 cells at 4 h after 2 Gy IR and CB-5083 treatment. Scale bars, 5 μm; n = 3.

(E) U2OS DR-GFP cells were either transfected with indicated siRNAs for 48 h or treated with 10 μM CB-5083 for 6 h. Cells were transfected with I-SceI plasmid for at least 8 h to induce DNA breaks, and HR repair was monitored by fluorescence-activated cell sorting (FACS).

(F) U2OS DR-GFP cells were transfected with I-SceI plasmid as in (E) and treated with p97 siRNA (10 nM over 48 h); n = 3; one-way ANOVA with Dunnett’s multiple comparisons. ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001; ^∗∗∗∗p < 0.0001 (E and F).

(G) Western blot showing RAD51 and p97 knockdown efficiencies.

(H) U2OS SA-GFP cells were treated as in (E); however, siRAD52 was used instead of siRAD51 as an internal control, and GFP positivity is a readout for SSA repair. The I-SceI group serves as a positive control that is normalized to 100%. One-way ANOVA with Dunnett’s multiple comparisons: ^∗p < 0.05; ^∗∗∗∗p < 0.0001; n = 4 replicates.

(I) Western blot showing knockdown of RAD52.

Data in (B) and (D) are presented as boxplot with interquartile range shown, and values beyond the 5th and 95th percentiles are shown as individual data points. The Mann-Whitney test was used for statistical analysis. NS, not significant; ^∗∗∗∗p < 0.0001.