Impact of CtIP depletion on repair frequencies in the presence or absence of hKU70.

<p><b>A</b>, Frequencies of Tus/<i>Ter</i>-induced repair in three independently derived <i>Ku70</i>^–/– 6x<i>Ter</i>-HR reporter clones (clones 27, 41, and 47) transiently expressing exogenous <i>hKU70</i> and transfected with siRNAs shown. Cells transiently co-transfected with empty pcDNA3beta or pcDNA3beta-<i>hKU70</i> expression vector and empty or 3xMyc-NLS Tus expression vectors treated with either si<i>LUC</i> or si<i>CtIP</i>. Each column represents the mean of duplicate samples from eight independent experiments (n = 8), values are corrected for transfection efficiency. Error bars: s.e.m. Tus-induced total HR, si<i>LUC</i> vs. si<i>CtIP</i> t test: #27 +EV, p = 0.0030; #41 +EV, p = 0.0207; #47 +EV, p = 0.0070; #27 +h<i>KU70</i>, p = 0.0047; #41 + h<i>KU70</i>, p = 0.0281; #47 + h<i>KU70</i>, p = 0.0148; Tus-induced STGC, t test: #27 +EV, p = 0.0011; #41 +EV, p = 0.0104; #47 +EV, p = 0.0070; #27 +h<i>KU70</i>, p = 0.0030; #41 + h<i>KU70</i>, p = 0.0207; #47 + h<i>KU70</i>, p = 0.0148; Tus-induced LTGC, t test: #27 +EV, p = 0.2786; #41 +EV, p = 0.5737; #47 +EV, p = 0.1304; #27 +h<i>KU70</i>, p = 0.8785; #41 + h<i>KU70</i>, p = 0.5737; #47 + h<i>KU70</i>, p = 0.5737; Tus-induced LTGC/(Total HR), t test: #27 +EV, p = 0.0002; #41 +EV, p = 0.0006; #47 +EV, p = 0.0006; #27 +h<i>KU70</i>, p = 0.0019; #41 + h<i>KU70</i>, p = 0.0030; #47 + h<i>KU70</i>, p = 0.0650; One-way ANOVA (Analysis of Variance) test comparing trend in Tus-induced Total HR: si<i>LUC</i> vs si<i>CtIP</i> all, p<0.0001; si<i>LUC</i> vs si<i>CtIP</i> +EV, p<0.0001; si<i>LUC</i> vs si<i>CtIP</i> +h<i>KU70</i>, p = 0.0002; si<i>LUC</i> all, p = 0.8904; si<i>CtIP</i> all, p = 0.1322. Tus-induced STGC, one-way ANOVA test: si<i>LUC</i> vs si<i>CtIP</i> all, p<0.0001; si<i>LUC</i> vs si<i>CtIP</i> +EV, p<0.0001; si<i>LUC</i> vs si<i>CtIP</i> +h<i>KU70</i>, p<0.0001; si<i>LUC</i> all, p = 0.9108; si<i>CtIP</i> all, p = 0.1155. Tus-induced LTGC, one-way ANOVA test: si<i>LUC</i> vs si<i>CtIP</i> all, p = 0.4334; si<i>LUC</i> vs si<i>CtIP</i> +EV, p = 0.3194; si<i>LUC</i> vs si<i>CtIP</i> +h<i>KU70</i>, p = 0.4144; si<i>LUC</i> all, p = 0.6254; si<i>CtIP</i> all, p = 0.2231. Tus-induced LTGC/(Total HR), one-way ANOVA test: si<i>LUC</i> vs si<i>CtIP</i> all, p<0.0001; si<i>LUC</i> vs si<i>CtIP</i> +EV, p = 0.0004; si<i>LUC</i> vs si<i>CtIP</i> +h<i>KU70</i>, p = 0.0012; si<i>LUC</i> all, p = 0.9449; si<i>CtIP</i> all, p = 0.2989. <b>B</b>, Frequencies of I-SceI-induced repair in three independently derived <i>KU70</i>^Δ/Δ 6x<i>Ter</i>-HR reporter clones. Cells transiently co-transfected with empty pcDNA3beta or pcDNA3beta-<i>hKU70</i> expression vector and empty or 3xMyc-NLS I-SceI expression vectors treated with either si<i>LUC</i> or si<i>CtIP</i>. Each column represents the mean of duplicate samples from eight independent experiments (n = 8), values are corrected for transfection efficiency. Error bars: s.e.m. I-SceI-induced total HR, si<i>LUC</i> vs. si<i>CtIP</i> t test: #27 +EV, p = 0.0379; #41 +EV, p = 0.0281; #47 +EV, p = 0.0499; #27 +h<i>KU70</i>, p = 0.0003; #41 + h<i>KU70</i>, p = 0.0019; #47 + h<i>KU70</i>, p = 0.0011; I-SceI-induced STGC, si<i>LUC</i> vs. si<i>CtIP</i> t test: #27 +EV, p = 0.0379; #41 +EV, p = 0.0281; #47 +EV, p = 0.0379; #27 +h<i>KU70</i>, p = 0.0002; #41 + h<i>KU70</i>, p = 0.0019; #47 + h<i>KU70</i>, p = 0.0011; I-SceI-induced LTGC, si<i>LUC</i> vs. si<i>CtIP</i> t test: #27 +EV, p = 0.1104; #41 +EV, p = 0.7984; #47 +EV, p = 0.3282; #27 +h<i>KU70</i>, p = 0.3282; #41 + h<i>KU70</i>, p = 0.1949; #47 + h<i>KU70</i>, p = 0.1949; I-SceI-induced LTGC/(Total HR), si<i>LUC</i> vs. si<i>CtIP</i> t test: #27 +EV, p = 0.0011; #41 +EV, p = 0.0379; #47 +EV, p = 0.0070; #27 +h<i>KU70</i>, p = 0.0006; #41 + h<i>KU70</i>, p = 0.0650; #47 + h<i>KU70</i>, p = 0.0070; I-SceI-induced Total HR, one-way ANOVA test: si<i>LUC</i> vs si<i>CtIP</i> all, p<0.0001; si<i>LUC</i> vs si<i>CtIP</i> +EV, p = 0.0139; si<i>LUC</i> vs si<i>CtIP</i> +h<i>KU70</i>, p<0.0001; si<i>LUC</i> all, p<0.0001; si<i>CtIP</i> all, p<0.0001. I-SceI-induced STGC, one-way ANOVA test: si<i>LUC</i> vs si<i>CtIP</i> all, p<0.0001; si<i>LUC</i> vs si<i>CtIP</i> +EV, p = 0.0106; si<i>LUC</i> vs si<i>CtIP</i> +h<i>KU70</i>, p<0.0001; si<i>LUC</i> all, p<0.0001; si<i>CtIP</i> all, p<0.0001. I-SceI-induced LTGC, one-way ANOVA test: si<i>LUC</i> vs si<i>CtIP</i> all, p<0.0001; si<i>LUC</i> vs si<i>CtIP</i> +EV, p = 0.1503; si<i>LUC</i> vs si<i>CtIP</i> +h<i>KU70</i>, p = 0.1010; si<i>LUC</i> all, p = 0.0010; si<i>CtIP</i> all, p<0.0001. I-SceI-induced LTGC/(Total HR), one-way ANOVA test: si<i>LUC</i> vs si<i>CtIP</i> all, p<0.0001; si<i>LUC</i> vs si<i>CtIP</i> +EV, p<0.0001; si<i>LUC</i> vs si<i>CtIP</i> +h<i>KU70</i>, p<0.0001; si<i>LUC</i> all, p = 0.0147; si<i>CtIP</i> all, p<0.0001. <b>C</b>, Observed repair frequencies for Tus or I-SceI induced HR, STGC and LTGC expressed as the ratio of si<i>CtIP</i> frequency/si<i>LUC</i> frequency for data from clones 27, 41 and 47 shown in panels <b>A</b> and <b>B</b>. One-way ANOVA test: Tus-induced total HR, p = 0.0779; Tus-induced STGC, p = 0.0564; Tus-induced LTGC, p = 0.2067. One-way ANOVA test: I-SceI-induced total HR, p<0.0001; I-SceI-induced STGC, p<0.0001; I-SceI-induced LTGC, p = 0.0832. <b>D</b>, RT qPCR analysis of <i>CtIP</i> mRNA in siRNA-transfected <i>Ku70</i>^–/–clones. Data normalized to <i>GAPDH</i> and expressed as fold difference from si<i>LUC</i> sample from the same experiment (x = -2^ΔΔCt, with ΔΔCt = [Ct_siCtIP-Ct_Gapdh]-[Ct_si<i>LUC</i>-Ct_{si<i>GAPDH</i>}]). Error-bars represent standard deviation of the ΔCt value (SDEV = √[SDEV_<i>CtIP</i>² + SDEV_<i>GAPDH</i>²]).