Loss of <i>Xrcc4</i> does not perturb HR regulation of Tus/<i>Ter</i>-induced STGC and LTGC.

<p><b>A</b>, Frequencies of Tus/<i>Ter</i>-induced repair <i>Xrcc4</i>^Δ/Δ clone 11 6x<i>Ter</i>-HR reporter clones stably transduced with pHIV-EV (lentiviral empty vector control) or with pHIV-<i>mXrcc4</i> (HA-tagged mouse Xrcc4 lentiviral expression vector) with selection of transduced cells in 100 μg/mL NTC. Cells were transiently co-transfected with empty or 3xMyc-NLS Tus expression vectors and siRNAs as shown. Each plot represents the mean of duplicate samples from eight independent experiments (n = 8). Error bars: s.e.m. <b><i>Xrcc4</i></b>^<b>Δ/Δ</b> <b>clone #11 pHIV-EV</b>: Tus-induced Total HR, t test: si<i>LUC</i> vs. si<i>BRCA1</i> p = 0.0005; si<i>LUC</i> vs. si<i>BRCA2</i> p<0.0001; si<i>LUC vs</i>. si<i>RAD51</i> p<0.0001; si<i>CtIP</i> vs. si<i>LUC</i> p<0.0001; si<i>SLX4</i> vs. si<i>LUC</i> p<0.0001. Tus-induced STGC, t test: si<i>LUC</i> vs. si<i>BRCA1</i> p<0.0001; si<i>LUC</i> vs. si<i>BRCA2</i> p<0.0001; si<i>LUC</i> vs. si<i>RAD51</i> p<0.0001; si<i>CtIP</i> vs. si<i>LUC</i> p<0.0001; si<i>SLX4</i> vs. si<i>LUC</i> p<0.0001. Tus-induced LTGC, t test: si<i>LUC</i> vs. si<i>BRCA1</i> p = 0.0153; si<i>LUC</i> vs. si<i>BRCA2</i> p = 0.1481; si<i>LUC</i> vs. si<i>RAD51</i> p = 0.0034; si<i>CtIP</i> vs. si<i>LUC</i> p = 0.2292; si<i>SLX4</i> vs. si<i>LUC</i> p = 0.0018. Tus-induced Ratio, t test: si<i>LUC</i> vs. si<i>BRCA1</i> p = 0.0001; si<i>LUC</i> vs. si<i>BRCA2</i> p = 0.0003; si<i>LUC</i> vs. si<i>RAD51</i> p<0.0001; si<i>CtIP</i> vs. si<i>LUC</i> p<0.0001; si<i>SLX4</i> vs. si<i>LUC</i> p<0.0001. <b><i>Xrcc4</i></b>^Δ<b>/</b>Δ <b>clone #11 pHIV-<i>mXrcc4</i></b>: Tus-induced Total HR, t test: si<i>LUC</i> vs. si<i>BRCA1</i> p = 0.0002; si<i>LUC</i> vs. si<i>BRCA2</i> p<0.0001; si<i>LUC</i> vs. si<i>RAD51</i> p<0.0001; si<i>CtIP</i> vs. si<i>LUC</i> p<0.0001; si<i>SLX4</i> vs. si<i>LUC</i> p<0.0001. Tus-induced STGC, t test: si<i>LUC</i> vs. si<i>BRCA1</i> p<0.0001;si<i>LUC</i> vs. si<i>BRCA2</i> p<0.0001; si<i>LUC</i> vs. si<i>RAD51</i> p<0.0001; si<i>CtIP</i> vs. si<i>LUC</i> p<0.0001; si<i>SLX4</i> vs. si<i>LUC</i> p<0.0001. Tus-induced LTGC, t test: si<i>LUC</i> vs. si<i>BRCA1</i> p = 0.0023; si<i>LUC</i> vs. si<i>BRCA2</i> p = 0.0240; si<i>LUC</i> vs. si<i>RAD51</i> p = 0.0002; si<i>CtIP</i> vs. si<i>LUC</i> p = 0.7398; si<i>SLX4</i> vs. si<i>LUC</i> p = 0.0022. Tus-induced Ratio, t test: si<i>LUC</i> vs. si<i>BRCA1</i> p = 0.0004; si<i>LUC</i> vs. si<i>BRCA2</i> p<0.0001; si<i>LUC</i> vs. si<i>RAD51</i> p<0.0001; si<i>CtIP</i> vs. si<i>LUC</i> p = 0.0049; si<i>SLX4</i> vs. si<i>LUC</i> p = 0.0051. <b>B</b>, Frequencies of I-SceI-induced repair <i>Xrcc4</i>^Δ/Δ clone 11 6x<i>Ter</i>-HR reporter clones stably transduced with pHIV-EV (lentiviral empty vector control, “EV”) or with pHIV-<i>mXrcc4</i> (HA-tagged mouse Xrcc4 lentiviral expression vector, “X4”) with selection of transduced cells in 100 μg/ml NTC. Cells were co-transiently transfected with empty, or 3xMyc-NLS I-SceI expression vectors and siRNAs as shown. Each plot represents the mean of duplicate samples from eight independent experiments (n = 8). Error bars: s.e.m. <b><i>Xrcc4</i></b>^Δ<b>/</b>Δ <b>clone #11 pHIV-EV</b>: I-SceI-induced total HR, t test: si<i>LUC</i> vs. si<i>BRCA1</i> p<0.0001; si<i>LUC</i> vs. si<i>BRCA2</i> p<0.0001; si<i>LUC</i> vs. si<i>RAD51</i> p<0.0001; si<i>CtIP</i> vs. si<i>LUC</i> p<0.0001; si<i>SLX4</i> vs. si<i>LUC</i> p<0.0001. I-SceI-induced STGC, t test: si<i>LUC</i> vs. si<i>BRCA1</i> p<0.0001; si<i>LUC</i> vs. si<i>BRCA2</i> p<0.0001; si<i>LUC</i> vs. si<i>RAD51</i> p<0.0001 si<i>CtIP</i> vs. si<i>LUC</i> p<0.0001; si<i>SLX4</i> vs. si<i>LUC</i> p<0.0001. I-SceI-induced LTGC, t test: si<i>LUC</i> vs. si<i>BRCA1</i> p = 0.3335; si<i>LUC</i> vs. si<i>BRCA2</i> p<0.0001; si<i>LUC</i> vs. si<i>RAD51</i> p<0.0001; si<i>CtIP</i> vs. si<i>LUC</i> p<0.0001; si<i>SLX4</i> vs. si<i>LUC</i> p = 0.0006. I-SceI-induced Ratio, t test: si<i>LUC</i> vs. si<i>BRCA1</i> p = 0.0001; si<i>LUC</i> vs. si<i>BRCA2</i> p = 0.0020; si<i>LUC</i> vs. si<i>RAD51</i> p<0.0001; si<i>CtIP</i> vs. si<i>LUC</i> p<0.0001; si<i>SLX4</i> vs. si<i>LUC</i> p = 0.6260. <b><i>Xrcc4</i></b>^Δ<b>/</b>Δ <b>clone 11 pHIV-<i>mXrcc4</i></b>: I-SceI-induced total HR, t test: si<i>LUC</i> vs. si<i>BRCA1</i> p<0.0001; si<i>LUC</i> vs. si<i>BRCA2</i> p<0.0001; si<i>LUC</i> vs. si<i>RAD51</i> p<0.0001; si<i>CtIP</i> vs. si<i>LUC</i> p<0.0001; si<i>SLX4</i> vs. si<i>LUC</i> p<0.0001. I-SceI-induced STGC, t test: si<i>LUC</i> vs. si<i>BRCA1</i> p<0.0001; si<i>LUC</i> vs. si<i>BRCA2</i> p<0.0001; si<i>LUC</i> vs. si<i>RAD51</i> p<0.0001; si<i>CtIP</i> vs. si<i>LUC</i> p<0.0001; si<i>SLX4</i> vs. si<i>LUC</i> p<0.0001. I-SceI-induced LTGC, t test: si<i>LUC</i> vs. si<i>BRCA1</i> p = 0.0001; si<i>LUC</i> vs. si<i>BRCA2</i> p = 0.0002; si<i>LUC</i> vs. si<i>RAD51</i> p<0.0001; si<i>CtIP</i> vs. si<i>LUC</i> p = 0.0590; si<i>SLX4</i> vs. si<i>LUC</i> p = 0.0001. I-SceI-induced Ratio, t test: si<i>LUC</i> vs. si<i>BRCA1</i> p = 0.0011; si<i>LUC</i> vs. si<i>BRCA2</i> p = 0.0330; si<i>LUC</i> vs. si<i>RAD51</i> p = 0.0491; si<i>CtIP</i> vs. si<i>LUC</i> p = 0.0017; si<i>SLX4</i> vs. si<i>LUC</i> p = 0.0136. <b>C</b>, RT qPCR analysis of <i>BRCA1</i>, BRCA2, <i>CtIP</i> and <i>SLX4</i> mRNA in siRNA-treated <i>Xrcc4</i>^Δ<i>/</i>Δ cells stably transduced with pHIV-EV (“EV”) or pHIV-<i>mXrcc4</i> (“X4”) derived lentivirus. 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 _target-Ct_Gapdh]-[Ct_si<i>LUC</i>-Ct_{si<i>GAPDH</i>}]). Error-bars represent standard deviation of the ΔCt value (SDEV = √[SDEV_{<i>TARGET</i>}² + SDEV_<i>GAPDH</i>²]). <b>D</b>, Western blot of RAD51 protein abundance in siRNA-treated stably transduced <i>Xrcc4</i>^Δ<i>/</i>Δ cells; pHIV-empty vector control (“EV”) or pHIV-<i>mXrcc4</i> (“X4”). <b>E</b>, Western blot of Brca1 protein abundance in siRNA-treated stably transduced <i>Xrcc4</i>^Δ<i>/</i>Δ cells; pHIV-empty vector control (“EV”) or pHIV-<i>mXrcc4</i> (“X4”).