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. Author manuscript; available in PMC: 2026 Aug 18.
Published in final edited form as: Cell Rep. 2026 Jul 15;45(7):117699. doi: 10.1016/j.celrep.2026.117699

Figure 5. SCLC cells expressing tyrosine phosphorylation-deficient RAD52 and RAD51 mutants exhibit decreased foci, increased TRCs and decreased DNA replication fork progression in response to DNA damage.

Figure 5.

(A) DMS53 cells expressing GFP-RAD52 wild type (WT) or GFP-RAD52 Y104 (YF) mutant were depleted of endogenous RAD52 using shRNA to the RAD52-UTR region. Cells plated onto the coverslips were treated with 4 Gy or without ionizing radiation (IR). Cells were incubated for 3 h after IR followed by immunofluorescence (IF) staining. RAD52-GFP (green) and DAPI-stained DNA (blue) were visualized with confocal microscope with 40× oil lens.

Representative images are shown (Single RAD52 foci images presented as black and white pseudo-color). The number of RAD52 foci per cell was plotted with GraphPad Prism (number of cells n = 123–139). Statistics was performed with a one-way ANOVA software: ns: not significant; **p = 0.0042 and ****p = 0.0001. Scale bars, 5 μm.

(B) RAD52 WT and RAD52 Y104F (YF) mutant DMS53 cells were treated with 4Gy or without IR. After treatment, cells were incubated for 4 h, then fixed for PLA (S9.6-PCNA). Scale bars, 10 μm. Quantification of PLA foci per nucleus in DMS53 cells expressing RAD52 WT and RAD52 Y104F without irradiation (113 and 164 nuclei, respectively) and RAD52 WT and RAD52 Y104F with 4Gy irradiation (147 and 104 nuclei, respectively). Data were analyzed with a one-way ANOVA: ns: not significant; *p value < 0.05 and ****p < 0.0001.

(C) Schematic representation of the DNA fiber assay. DMS53 cells harboring Flag-tagged RAD51-WT or RAD51-YF (Y315F) mutant were depleted of endogenous RAD51 with shRNA to the RAD51-UTR regionCells were subjected to sequential labeling with thymidine analogs CldU (50 μM) and IdU (100 μM), respectively, for the indicated time periods. For HU treatment, cells were exposed to HU (2 mM) together with IdU.

(D) Representative images of RAD51-WT or RAD51-YF labeled with CIdU and IdU in the presence or absence of HU treatment. Scale bars, 5 μm.

Quantification of DNA Fiber length described in (A). Number of fibers counted n = 50. Statistics were performed with a one-way ANOVA: ****p < 0.0001; ns: not significant.

Data in A, B, and D represented as mean +/− SEM.