Figure 4.

SPOC1-expression levels modulate NHEJ activity. (A) siRNA-mediated SPOC1 depletion increases NHEJ activity in H1299.EJ reporter cells compared to control siRNA-transfected cells as quantified by flow cytometry detecting EGFP expression. NHEJ activity is only evident after I-Scel-mediated cleavage in cells transfected with a plasmid expressing cytoplasmic SceI fused to glucocorticoid receptor ligand-binding domain (SceI-GR), plus TA to translocate the fusion protein into the nucleus. (B) Enhanced expression of SPOC1wt and a SPOC1 N-terminal domain deletion (deltaN), but not a PHD-deficient derivative (PHDmt) strongly reduced NHEJ activity when analyzed and quantified as in (A). (C) DNA repair activity by HR in H1299.GC reporter cells after SPOC1 siRNA or control siRNA transfection, quantified as in (A), were similar. (D) HR-mediated DNA repair activity in U2OS-DR-GFP reporter cells was reduced by >60% after siRNA-induced SPOC1 knockdown compared to control siRNA treated cells. Knockdown of CtIP, a protein critical for the initial steps of HR, reduced HR activity by ∼90% compared to the control siRNA transfected cells. All data are from triplicates derived from three independent experiments (±SEM) ****P ≤ 0.0001. (A–D) Successful SPOC1 and CtIP knockdown as well as equivalent expression levels of SPOC1 wt and mutant proteins were confirmed by immunoblotting of cell extracts using anti-SPOC1, anti-CtIP and anti-GAPDH antibodies.