Fig. 4: RPA:RAD52:MRE11 suppression reduces the toxicity of Polθ depletion. | Nature Communications

Fig. 4: RPA:RAD52:MRE11 suppression reduces the toxicity of Polθ depletion.

From: Mechanisms of synthetic lethality between BRCA1/2 and 53BP1 deficiencies and DNA polymerase theta targeting

Fig. 4

a Survival after control, Polθ siRNA, or Polθ siRNA with RPA siRNA. n = 3 biological replicates. b FLAG-RAD52 foci after control or Polθ siRNA with and without RPA siRNA. n = 100 cells from 2 biological replicates, per condition. c Breaks and gaps / metaphase after control (−) or Polθ (+) siRNA with or without RPA siRNA. N ≥60 metaphases from 3 biological replicates. d Survival of cells infected with empty (−), WT-RAD52 and aa 254–286 deleted RAD52 mutant retroviruses each resistant to RAD52 siRNA, treated with Polθ siRNA and 8 nM RAD52 (+) or control siRNA (−). n = 4 biological repeats. e Survival after Polθ siRNA and MRE11 inhibitor, mirin. n = 3 biological repeats. f FLAG-RAD52 foci after treatment with vehicle or 2.5 μM mirin. n = 3 biological repeats. g Proximity-linked ligation assay foci (PLA) between MRE11 and BrdU, treated with indicated siRNA and/or inhibitors for 72 h and with 10 µM BrdU 48 h before fixation. n = 100 cells from 2 biological replicates. h IdU:CIdU ratios from MEFS treated with Polθ siRNA, and/or RAD52 inhibitor, 6-OHD and hydroxyurea (5 mM, 3 h). n > 350 fibres from 3 replicates. i Native BrdU tracts after control or Polθ siRNA and 0.15 µM RAD52 inhibitor 6-OHD and/or 5 µM mirin for 72 h. n ≥ 1400 tracks from 3 biological replicates. j PRR assay of MEFs with Polθ or BRCA1 siRNA and Polθ siRNA with and without RAD52 inhibitor, 6-OHD. n > 25 from 3 biological replicates. k PRR assay of CAL51 with Polθ or BRCA1 siRNA and Polθ siRNA with and without RAD52 inhibitor, 6-OHD. n > 25 from 3 biological replicates. l γH2AX foci after treatment with Polθ siRNA and RAD52 inhibitor 6-OHD or MRE11 inhibitor, mirin. n = 100 cells from 2 biological repeats. For a–g and j–l, data shown are mean ± SEM. For h and l, data shown are median. Statistical analysis in a–g and j–l was performed using a two-tailed Student’s t-Test, without adjustment for multiple comparisons. Statistical analysis in h and i was performed using a Mann–Whitney test. Source data are provided as a Source Data file.

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