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. Author manuscript; available in PMC: 2022 Feb 18.
Published in final edited form as: Nature. 2021 May 12;594(7862):283–288. doi: 10.1038/s41586-021-03538-8

Extended Data Fig. 5 |. Regulation of RAD51AP1 localization to DSBs.

Extended Data Fig. 5 |

a, U2OS cells carrying an array of tetracycline responsive elements (TREs) were transfected with a plasmid expressing the TetR-VP16-KillerRed (TA-KR) fusion protein. The TA-KR fusion protein binds the TRE array and induces DSBs upon light activation. The formation of RAD51AP1 or RAD51 foci at the TA-KR-marked locus was analysed by immunostaining. Scale bars, 10 μm. b, Immunostaining of RAD51AP1 in cells that were not irradiated or were irradiated with 2 Gy IR. Cells were analysed 2 h after IR. Scale bars, 10 μm. c, Correlation between the numbers of RAD51AP1 and RPA32 foci in IR-treated cells as determined by linear regression. The numbers of RAD51AP1 and RPA32 foci were quantified in individual cells (n = 260 cells analysed in one experiment). Individual cells were plotted according to the numbers of RAD51AP1 and RPA32 foci in them. d–g, Asynchronously growing U2OS cells were treated with or without DRB for 4 h, exposed to 2 Gy IR or mock-treated, and analysed in 2 h. d, Immunostaining of RAD51AP1 and γH2AX. Scale bars, 10 μm. e, Numbers of RAD51AP1 foci in individual cells were plotted as mean ± s.d. (n = 401 cells for no IR, n = 408 cells for +IR, and n = 408 cells for +IR +DRB, analysed in one experiment). f, Numbers of γH2AX foci in individual cells were plotted as mean ± s.d. (n = 313 cells for − DRB and n = 294 cells for +DRB, analysed in one experiment). g, Numbers of RAD51AP1 foci in PCNA+ cells were plotted as mean ± s.d. (n = 360 cells for −DRB and n = 303 cells for +DRB, analysed in one experiment). h, i, U2OS cells transfected with control, UAF1 (h) or CtIP (i) siRNA were irradiated with 2 Gy IR. Immunostaining of RAD51AP1 was done 2 h after IR. Numbers of RAD51AP1 foci in individual cells were plotted as mean ±s.d. ***P < 0.001 (two-sided Student’s t test; P < 0.0001 in h, i. h, n = 483 cells for siCTRL, n = 527 cells for siUAF1 analysed in one experiment. i, n = 200 cells for siCTRL, n = 182 cells for siCtIP analysed in one experiment.