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. 2005 Jun 2;24(13):2447–2457. doi: 10.1038/sj.emboj.7600704

Figure 3.

Figure 3

ASCIZ is required for Rad51 focus formation and cell survival in response to MMS treatment. (A) Western blot analysis of ASCIZ, Rad51 and actin from ASCIZ siRNA- and luciferase siRNA-treated U2OS cells. (B) Apoptosis in U2OS cells siRNA-treated as in (A) at the indicated times after 0.005% MMS addition (mean±s.e. of three independent experiments), detected by TUNEL staining. *P<0.01 (paired t-test, two-sided) versus mock and GL2 at 24 h. (C) MMS-induced Rad51 focus formation in siRNA-treated U2OS cells. The bottom panel shows enlargements of nuclei labeled by arrows above. (D) Quantitation of Rad51 focus formation (mean±s.e. of 2–3 independent experiments) in siRNA-treated U2OS cells under basal conditions and in response to MMS or IR as in (B) and (E). Similar results for another ASCIZ siRNA are shown in Supplementary Figure S6C. (E) γH2AX formation in siRNA-treated U2OS cells with or without MMS treatment. The right panel shows an overexposed Western blot demonstrating the specificity of the antibody. In the experiment shown here, 0.14 and 99.5% of control and 0.25 and 97.1% of ASCIZ-depleted cells contained γH2AX in the absence or presence of MMS treatment, respectively (>170 nuclei scored per sample). (F) Formation of Rad51 foci but not GFP-ASCIZ foci in response to IR (left panels), and normal Rad51 focus formation after IR in ASCIZ siRNA-treated U2OS cells (right panels).