Figure 1.

Absence of c-Abl kinase activity results in better DSB rejoining. (a) HeLa cells were pretreated with STI-571 and γ-irradiated. c-Abl tyrosine autophosphorylation was assayed by immunoprecipitation of endogenous c-Abl, followed by WB for the presence of phosphorylated tyrosine. (b) wt MEFs were pretreated with STI-571, γ-irradiated and subjected to the comet assay following 24 h of post-IR incubation at 37°C. The comet moment value represents the extent of genomic DNA fragmentation in individual cells (N=50). (c) c-Abl−/− MEFs reconstituted for c-Abl expression were γ-irradiated, harvested after 24 h of post-IR incubation and subjected to the comet assay. Data are expressed as mean±S.D., N=50 (left panel). Presence of c-Abl in the reconstituted cells and not in non-reconstituted c-Abl−/− cells was verified by WB (right panel). (d) HeLa cells were pretreated with STI-571 and γ-irradiated, cells were collected after 24 h of incubation at 37°C and subjected to PFGE. (e) Same as in (d), the amount of residual DNA fragmentation was quantified and the ratio of fragmentation between untreated and STI-571-treated cells was plotted. Error bars represent S.E.M. for seven independent experiments. The P-value for the paired values of STI-571-treated and untreated cells was calculated using Wilcoxon's signed-rank test, P-value <0.001. (f) HeLa cells were transiently transfected with a pSuper construct carrying shRNA to target c-Abl¹² or with an empty vector. Following puromycin semiselection, the cells were γ-irradiated and analyzed by PFGE (left panel). Error bars represent SEM for four independent experiments. The P-value for the paired values of knockdown and control cells was calculated using Wilcoxon's signed-rank test, P-value <0.001. Efficient knockdown of c-Abl was verified by WB (right panel)