Fig 2: Loss of ATM delays DSB repair kinetics of siDAB2IP cells. (A) All sublines were exposed to γ-rays of 2 Gy and immunostained for γH2AX (green) foci at 0, 2, 8 and 24 h post-irradiation (average 100 nuclei; magnification ×40). (B) DNA repair kinetics in the four sublines are presented as a plot of the percentage of remaining foci against time. The results are presented as the mean ± standard error of the mean of triplicate experiments. ATM, ataxia-telangiectasia mutated; DSB, double-strand break; DAB2IP, disabled homolog 2 interactive protein; DNA-PKcs, catalytic subunit of DNA-dependent protein kinase; si, small interfering RNA.

Fig 3: Loss of ATM delays DSB repair kinetics of siDAB2IP cells. (A) Distribution of NF-κB (green) was determined using an immunofluorescence assay (magnification ×100). DAPI (blue) was utilized to mark the location of nuclei. Above, NF-κB distributed in the cytoplasm only was determined as negative. Below, NF-κB translocated into the nucleus was determined as positive. (B) All sublines were irradiated with 2 Gy and immunostained for NF-κB at 2 h post-IR (magnification ×40). (C) Percentage of nuclear NF-κB (+) cells against time were plotted (average 100 nuclei). The results are presented as the mean ± standard error of the mean of triplicate experiments. *P<0.05, compared with the siControl cells; **P<0.05, compared with the siDAB2IP cells. ATM, ataxia-telangiectasia mutated; DSB, double-strand break; DAB2IP, disabled homolog 2 interactive protein; DNA-PKcs, catalytic subunit of DNA-dependent protein kinase; si, small interfering RNA; NF-κB, nuclear factor-κB; DAPI, 4′,6-diamidino-2-phenylindole.

Fig 4: Loss of ATM enhances sensitivity of DAB2IP-deficient cells to γ-ray. (A) Cells were transfected with different siRNA as indicated. The expression levels of DAB2IP, ATM and DNA-PKcs in the sublines were detected using western blot analysis. GADPH was loaded as an internal control. (B) All sublines were subjected to increasing doses of radiation (0, 2 and 5 Gy). The colonies were counted and the surviving fraction was calculated following 14 days of incubation. The results are presented as the mean ± standard error of the mean of triplicate experiments. *P<0.05, compared with the 5637 siDAB2IP cells; **P<0.05, compared with the 5637 siDAB2IP cells. ATM, ataxia-telangiectasia mutated; DAB2IP, disabled homolog 2 interactive protein; DNA-PKcs, catalytic subunit of DNA-dependent protein kinase; si, small interfering RNA.

Fig 5: Mechanical tension of the actin cytoskeleton is not necessary to sustain DAB2IP protein levels in confluent cells. (A,B) FAK inhibition reduces DAB2IP protein levels in subconfluent cells, but not in confluent monolayers. MCF10A were seeded and cultured for 48 h to a subconfluent, sparse or confluent condition, as in Figure 1, and then treated with 10 µM PF573228 (PF573) for 6 h before lysis. DAB2IP, p-FAK Y397, total FAK and E-cadherin were detected by immunoblotting, with HSP90 as loading control. (C–E) Disruption of actin cytoskeleton reduces DAB2IP protein levels in subconfluent cells, but not in confluent monolayers. MCF10A were seeded and cultured to subconfluent or confluent conditions, and then were exposed to Cytochalasin D (Cyt D, 10 µM for 4 h), Latrunculin A (LatA, 0.5 µM for 4 h), or Blebbistatin (Bleb, 50 µM for 24 h); then, cells were processed for immunofluorescence or Western blot. (C) Representative images of treated and control cells. F-actin was visualized with rhodamine phalloidin (red). Nuclei were labeled with Hoechst (blue). Scale bar = 20 µm. (D,E) DAB2IP was analyzed by immunoblotting after cells were treated with the indicated drugs in subconfluent conditions (D) or as a confluent monolayer (E). HSP90 was blotted as a loading control.