Loss of Ku70 can diminish the influence of Fanca on Alt-EJ, but not cisplatin sensitivity.

<p>(<b>A</b>) Transient expression of Fanca in <i>Fanca^−/−</i> mES cells promotes Alt-EJ but not Distal-EJ. The EJ2-GFP and EJ5-GFP reporters were integrated into <i>Fanca^−/−</i> mES cells. Shown are RT-PCR signals confirming the exon 37–39 deletion in <i>Fanca^−/−</i>, which causes a frame shift at N1202. These cells were transfected with expression vectors for I-SceI along with either mouse Fanca or empty vector (EV). Repair values (%GFP+) from these transfections are shown relative to parallel EV. *<i>P</i><0.0001 (<i>n</i> = 9). Also shown are immunoblot signals detecting expression of Fanca from cells transfected with the expression vector, although this anti-human FANCA antibody is unable to detect endogenous Fanca in WT (†). (<b>B</b>) Generation of <i>Ku70</i> frame-shift mutations in <i>Fanca^−/−</i> EJ2-GFP mES cells. Shown is the predicted target cut site in <i>Ku70</i> exon 5 for a guide RNA and CAS9, along with sequence of the <i>Ku70</i> frame-shift mutations (13 and 2 nt deletions, Δ13 and Δ2, respectively) that resulted from two rounds of CAS9 expression. Shown are immunoblot signals of these <i>Fanca^−/−Ku70^−/−</i> mES cells for Ku70 and Fanca, following transient transfection with Ku70 or Fanca expression vectors, respectively. (<b>C</b>) Loss of Ku70 diminishes the influence of Fanca on Alt-EJ. The <i>Fanca^−/−</i> and <i>Fanca^−/−Ku70^−/−</i> EJ2-GFP cell lines were co-transfected with Fanca, Ku70, or EV and I-SceI expression vectors. Shown are %GFP+ frequencies normalized to transfected cells using transfection with a GFP expression vector in parallel. *<i>P</i><0.0001 (<i>n</i> = 11). On the right are the fold changes in GFP+ frequencies relative to EV. *<i>P</i><0.0001 (<i>n</i> = 11). (<b>D</b>) Loss of <i>Ku70</i> causes cisplatin and IR hypersensitivity in <i>Fanca-/-</i> cells. WT, <i>Fanca^−/−</i>, <i>Fanca^−/−Ku70^−/−</i>, and <i>Ku70^−/−</i> mES cell lines were treated continually with cisplatin, or exposed to IR (Cs137), and cultured to allow colony formation. Shown is fraction clonogenic survival relative to untreated cells (UN).