Figure 4.
CAM833 inhibits RAD51 molecular clustering at DNA damage sites visualized by SMLM
(A) Diagrammatic representation of the biallelic truncating mutations (red and orange) affecting BRCA2 in the patient-derived cell line EUFA423, and their functional complementation by full-length BRCA2 (green) in EUFA423 + BRCA2 cells. Black vertical lines depict the approximate positions of the BRC repeats.
(B) Distribution of the number of RAD51 molecules contained within damage-induced clusters in EUFA423 or EUFA423 + BRCA2 cells, without or with exposure to 25 μM CAM833, 3 h after exposure to 3 Gy ionizing radiation. The box plot was generated using the Matlab boxplot function. The central mark indicates the median (purple dot). The bottom and top edges of the box indicate the 25th and 75th percentiles, respectively. The whiskers extend to the most extreme data points not considered outliers. The mean is marked by a purple line. ∗∗∗ and n.s. indicates p values lower than 10−5 and not significant differences, respectively.
(C) Representative SMLM images of RAD51, represented as 2D Voronoi polygons. The color of the polygons shows molecular densities normalized to the maximum value. Scale bar, 500 nm.
(D) High-magnification SMLM images of damage-induced RAD51 filaments in EUFA423 + BRCA2 cells (DMSO-control left-hand panels), and their suppression by CAM833 (right-hand panels), under the same experimental conditions, at higher magnification. Scale bar, 200 nm. Images are shown either at low-resolution (LR) or super-resolved (SR). Results are representative of two independent repeats.