Figure 4. Effects of ZRANB3 depletion in mammalian cells.

(A) Cell competition assay in U2OS cells treated with control siRNAs or three independent ZRANB3 siRNAs following treatment with camptothecin (CPT, 5 nM), hydroxyurea (HU, 2 mM) or cisplatin (CIS, 0.5 μM). The data represent the average and standard deviation of three independent experiments.

(B) Cell competition assay in U2OS cells expressing either control or SMARCAL1 shRNAs treated with control or ZRANB3 siRNAs following treatment with camptothecin (CPT, 5 nM). Error bars have been calculated as in (A).

(C) Graphical representation of the frequencies of sister chromatid exchanges (SCEs) of mitotic chromosomes isolated from U2OS cells transfected with control or ZRANB3 siRNA with or without mitomycin C (MMC, 20 nM) or camptothecin (CPT, 2.5 nM) treatment. The SCE frequencies of U2OS cells expressing a ZRANB3 cDNA clone resistant to ZRANB3 siRNA treatment are indicated. The average frequencies of SCEs and the standard deviation are indicated. Statistically significant p-values calculated using the Mann-Whitney test are indicated by asterisks (^*p<0.05, ^***p<0.001). n.s., not significant.

(D) Chromosome spreads from U2OS cells transfected with control or ZRANB3 siRNA after camptothecin (CPT, 2.5 nM) treatment. SCEs are indicated.

(E) Percentage of U2OS cells transfected with control or ZRANB3 siRNAs displaying more than 10 RAD51 foci. Cells were fixed 6 h or 12 h following 1 h camptothecin (CPT, 10 nM) treatment. The percentage of U2OS cells with more than 10 RAD51 foci that expressed siRNA resistant cDNA clones coding for either WT, PIP and APIM or NZF mutant ZRANB3 is also indicated. The data represent the average and standard deviation of three independent experiments in which 100 or more cells were counted.