FIGURE 4.

Knockdown of filamin-A inhibits homologous recombinational repair of DSB. The filamin-A expression in HT1080–1885 cells, which host an HR assay substrate, was knocked down by shRNA. Five control clones (labeled as 1–5) and seven knockdown clones (labeled as A-G) were isolated. Break-induced HR was measured in these clones (See Results for a description of the HR assay). Panel A shows the level of filamin-A protein levels in control and knockdown clones. Clones 1–5 are cells expressing a control shRNA, and clones A-G are cells expressing filamin-A specific shRNA. HA-ISceI blot (bottom panel) shows the expression level of the I-SceI enzymes that introduces the site specific DSB in the HT1080–1885 cells. Panel B illustrates the recombination substrate and the HR mechanism resulting in the reconstitution of a functional puromycin resistance gene. Panel C shows the HR frequencies of individual clones. Shown are means and standard deviations of 6–8 experiments for each clone. Panel D summarizes HR frequencies for the filamin-A knockdown clones and the control clones. Shown are the means and standard deviations. The p-value indicates the statistical significance of the difference between means of the control clones and the filamin-A knock down clones (t-test).