Dipeptidyl peptidase 9 triggers BRCA2 degradation and promotes DNA-damage repair (2022)
Figure 6

Figure 6: DPP9-deficient cells show defects in HR repair DPP9 and form fewer RAD51 foci upon MMC treatment

A. Graphical presentation of the DR-GFP reporter assay for HR. The construct includes two mutated GFP genes (SceGFP and iGFP) oriented as direct repeats. The SceGFP gene is mutated to contain the recognition site for the rare-cutting endonuclease I-SceI and a STOP codon, while the iGFP is a truncated form of GFP. Cells expressing the DR-GFP are transfected with I-SceI, that forms a DSB within the SceGFP. For HR-mediated repair, the iGFP serves as template for HR-mediated repair of the DSB in GFP (Pierce et al, 1999).

B. DPP9-silenced cells are less efficient in repair of DSBs by HR. HCT116 cells stably expressing the DR-GFP reporter assay for HR efficiency were transiently transfected with the indicated siRNAs, and transfected with an I-SceI-expression vector. 48 h after transfection, the percentage of GFP-positive cells was measured using flow cytometry analysis as an indication for HR efficiency. Graph shows the mean ± SEM from seven biological replicates. Data were analysed by a paired two-tailed t-test (** = p ≤ 0.01, *** = p ≤ 0.001, **** = p ≤ 0.0001).

C. Representative PLA images of BRCA2-PALB2 PLA experiments showing more PLA events (white) in HeLa cells silenced for DPP9 (siDPP9) with respect to cells treated with non-targeting siRNA (siNT). Phalloidin (green) stains actin filaments, DAPI (blue) stains the nucleus. Scale bar 10 µm. Anti-PALB2: RRID:AB_890607, anti-BRCA2: RRID:AB_2259370.

D. Quantification of PLAs experiments showing more BRCA2-PALB2 PLA events in cells silenced for DPP9 (siDPP9) in comparison to non-targeting controls (siNT). Each dot represents the number of PLA events in a single cell, from four biological replicates. The technical control samples (NgtCntrl) omitted the BRCA2 antibody. The number of foci is shown based on their cellular localisation. Data were analysed by a two-way ANOVA, with Tukey's Multiple Comparison test. Shown are Mean ± SEM (**** = p ≤ 0.0001).

E. Representative PLA images of γH2Ax-BRCA2 PLA experiment showing a reduction in the number of PLA events (white) between γH2Ax and BRCA2 in HeLa DPP9 KD cells. Phalloidin (green) stains actin filaments, DAPI (blue) stains the nucleus. Scale bar 10 µm. Anti-γH2Ax: RRID:AB_2118009, anti-BRCA2: RRID:AB_2259370 (these images, along with the corresponding controls can be found in Appendix Figure S1D).

F. Quantification of PLAs showing fewer MMC-induced γH2Ax-BRCA2 PLA events in HeLa DPP9 KD cells, in comparison to HeLa WT cells. Each dot represents the number of PLA events in a single cell, from three biological replicates. The technical control samples (NgtCntrl) omitted the γH2Ax antibody. Data were analysed by a two-way ANOVA, with Tukey's Multiple Comparison test. Shown are Mean ± SEM (**** = p ≤ 0.0001).

G. Graph showing fewer RAD51 foci in DPP9-silenced cells compared to control cells, following exposure to MMC. Each dot represents the number of RAD51 foci in a single cell, from two to six biological replicates: siNT-MMC (n = 6), siNT+MMC (n = 6), siDPP9-MMC (n = 5), siDPP9+MMC (n = 5), siBRCA2-MMC (n = 3), siBRCA2+MMC (n = 3), siRAD51-MMC (n = 2), siRAD51+MMC (n = 2). Data were analysed by a two-way ANOVA, with Tukey's Multiple Comparison test. Shown are Mean ± SEM (**** = p ≤ 0.0001).