Extended Data Fig. 10: Effects of BRCA2 depletion and BRCA2-RAD51 interruption on PARPi-induced PARP1 retention.
From: BRCA2 prevents PARPi-mediated PARP1 retention to protect RAD51 filaments
(a) Experimental schematic depicting U2OS cells NT and 0.1 µM CPT treated cells were pulse-labeled with 10 µM EdU for 30 min followed by PARPi treatment at the mature phase of recombinase assembly. (b-d) Quantification of levels of RAD51, PARP1 and BRCA2 at naDNA over 5 h of recovery in U2OS cells WT, compared to (e,f) U2OS cells lacking BRCA2 expression (siBRAC2), in presence and absence of PARPi. (g) Representative super-resolution images of a single nucleus, post damage, and stained for naDNA (yellow), PARP1 (red) and RAD51 (cyan). Scale bar = 2 μm. Western blots showing siRNA-mediated knockdown of BRCA2 in U2OS cells. Stain free gel used as a loading control. (h) Experimental schematic depicting U2OS cells in media containing CAM833 followed by PARPi treatment at the mature phase of recombinase assembly. Cartoon showing CAM833 disrupting the protein-protein interaction between RAD51 and BRCA2. High-magnification SMLM images of damage induced RAD51 filament (cyan) in U2OS cells (left) and their suppression by CAM833 (right) at naDNA (yellow), under the same experimental conditions. Scale bar = 0.2 μm. (i,j) Levels of RAD51 and PARP1 at naDNA over 5 h of recovery in U2OS cells with CAM833 in media. CAM833 potentiates toxicity and synergizes with PARPi leading to PARP1 retention. We note that for both RAD51 and PARP1 the CPT only and CPT with PARPi data plotted in this panel is also used in panels e,f for comparison. Quantification is from two independent experiments and values on graph indicate P-values of unpaired two-sample t-tests between each sample set. (k,l) Representative super-resolution images of a single nucleus, post damage, and stained for naDNA (yellow), PARP1 (magenta) and RAD51 (cyan) in presence and absence of PARPi. Scale bar = 2 μm. Cartoons were created in BioRender. Lahiri, S. (2025) https://biorender.com/q21l624. *P < 0.05, **P < 0.01, and ***P < 0.001 and ****P < 0.0001. CPT = 0.1 µM | [CAM833] = 25 µM.
