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. 2021 Jun 18;81(16):4319–4331. doi: 10.1158/0008-5472.CAN-20-3651

Figure 4.

Figure 4. CIP2A is an interacting partner of TopBP1 and promotes mitotic progression of DNA damaged cells. A, Schematic presentation of breast cancer cell line cDNA fragments coding for TopBP1 domains that interact with full length CIP2A in a yeast two-hybrid assay. Numbers in the TopBP1 drawing refer to BRCT domains 1–8. AAD, ATR activation domain. Analysis of the minimal common overlapping region between the TopBP1 fragments interacting with CIP2A reveal the TopBP1 aa. 829–853 as a candidate CIP2A interaction domain. B, Coimmunoprecipitation of endogenous CIP2A and γH2AX in HEK293 cells transiently overexpressing GFP or full-length TopBP1-GFP as indicated. Input 5% of total IP. C, Coimmunoprecipitation of CIP2A in HEK293 cells transiently overexpressing V5-tagged CIP2A and GFP-tagged empty vector (EV) or TopBP1 truncated mutants T0, T1, T2, T3 as indicated in D. Input 5% of total IP. D, Schematic representation of TopBP1 mutants used in B and C. Relative interaction efficiencies are estimated from the experiment, where all indicated mutants were included. E, Immortalized MCF10A cells transfected with nontargeting (SCR) or CIP2A siRNAs for 48 hours. Immunoblot of whole-cell extracts probed for pATR, total ATR, and CIP2A. Vinculin was used as a loading control. Relative quantifications of pATR/ATR and CIP2A plotted as mean± SD from five replicates. F, MDA-MB-231 cells transfected with nontargeting (SCR) and CIP2A targeting siRNAs for 72 hours and overexpressing TopBP1 mutants T0 and T1 as indicated for 48 hours. Immunoblot of whole-cell extracts probed for pATR, γH2AX, and CIP2A. Actin was used as a loading control. Relative quantifications of γH2AX plotted as mean± SD from two replicates. G, IR-induced TopBP1 foci formation in MCF10A cells transfected with SCR or CIP2A siRNA as indicated for 48 hours. Cells were treated with 5 Gy radiation for 1 hour and stained for CIP2A or TopBP1. H, Quantifications of the nuclear foci from G expressed as mean ± SD from representative experiment of three experiments with similar results I, IR-induced RAD51 foci formation in mouse mammary epithelial cells (MMEC) isolated from WT and Cip2a−/− mice cultured in vitro for 48 hours, treated with 5 Gy radiation for 2 hours. J, Quantifications of the foci expressed as mean ± SD of representative experiment. G–J, Images were taken at ×63 on 3i spinning disk confocal and at least 150 cells quantified per each condition using speckle counter pipeline on CellProfiler; scale bar, 10 μm. E–J, All statistical analyses were conducted with the Welch Student t test for unequal variances; *, P < 0.05; **, P < 0.01. K, Schematic presentation of the role of CIP2A in directly inhibiting TopBP1/RAD51-elicited G2–M checkpoint activation in nontransformed mammary epithelial cells.

CIP2A is an interacting partner of TopBP1 and promotes mitotic progression of DNA damaged cells. A, Schematic presentation of breast cancer cell line cDNA fragments coding for TopBP1 domains that interact with full length CIP2A in a yeast two-hybrid assay. Numbers in the TopBP1 drawing refer to BRCT domains 1–8. AAD, ATR activation domain. Analysis of the minimal common overlapping region between the TopBP1 fragments interacting with CIP2A reveal the TopBP1 aa. 829–853 as a candidate CIP2A interaction domain. B, Coimmunoprecipitation of endogenous CIP2A and γH2AX in HEK293 cells transiently overexpressing GFP or full-length TopBP1-GFP as indicated. Input 5% of total IP. C, Coimmunoprecipitation of CIP2A in HEK293 cells transiently overexpressing V5-tagged CIP2A and GFP-tagged empty vector (EV) or TopBP1 truncated mutants T0, T1, T2, T3 as indicated in D. Input 5% of total IP. D, Schematic representation of TopBP1 mutants used in B and C. Relative interaction efficiencies are estimated from the experiment, where all indicated mutants were included. E, Immortalized MCF10A cells transfected with nontargeting (SCR) or CIP2A siRNAs for 48 hours. Immunoblot of whole-cell extracts probed for pATR, total ATR, and CIP2A. Vinculin was used as a loading control. Relative quantifications of pATR/ATR and CIP2A plotted as mean± SD from five replicates. F, MDA-MB-231 cells transfected with nontargeting (SCR) and CIP2A targeting siRNAs for 72 hours and overexpressing TopBP1 mutants T0 and T1 as indicated for 48 hours. Immunoblot of whole-cell extracts probed for pATR, γH2AX, and CIP2A. Actin was used as a loading control. Relative quantifications of γH2AX plotted as mean± SD from two replicates. G, IR-induced TopBP1 foci formation in MCF10A cells transfected with SCR or CIP2A siRNA as indicated for 48 hours. Cells were treated with 5 Gy radiation for 1 hour and stained for CIP2A or TopBP1. H, Quantifications of the nuclear foci from G expressed as mean ± SD from representative experiment of three experiments with similar results I, IR-induced RAD51 foci formation in mouse mammary epithelial cells (MMEC) isolated from WT and Cip2a−/− mice cultured in vitro for 48 hours, treated with 5 Gy radiation for 2 hours. J, Quantifications of the foci expressed as mean ± SD of representative experiment. G–J, Images were taken at ×63 on 3i spinning disk confocal and at least 150 cells quantified per each condition using speckle counter pipeline on CellProfiler; scale bar, 10 μm. E–J, All statistical analyses were conducted with the Welch Student t test for unequal variances; *, P < 0.05; **, P < 0.01. K, Schematic presentation of the role of CIP2A in directly inhibiting TopBP1/RAD51-elicited G2–M checkpoint activation in nontransformed mammary epithelial cells.