FIG 8.

Nbs1 knockdown disrupts MRN complex formation. (A) Whole-cell lysates were harvested from HFK, CIN612, and CIN612 9E cells stably expressing shScramble or shNBS1 at T₀ and after 72 h of differentiation in high-calcium medium (Ca). Immunoblotting was performed using Mre11, Rad50, and Nbs1 antibodies. GAPDH was used as a loading control. (B) Total, nuclear (Nuc), and cytoplasmic (Cyto) lysates were harvested from HFK and CIN612 cells. Immunoblotting was performed using Mre11, Rad50, and Nbs1 antibodies. (C) Total, nuclear (Nuc), and cytoplasmic (Cyto) lysates were harvested from stable CIN612 9E shScramble and CIN612 9E shNBS1 cells. Immunoblotting was performed using Mre11, Rad50, and Nbs1 antibodies. For panels B and C, lamin A/C and tubulin were used to confirm nuclear and cytoplasmic fractionation, respectively. (D) DNA was harvested from CIN612 9E cells at T₀ and after 72 h of differentiation in high-calcium medium with dimethyl sulfoxide (DMSO) as a vehicle control or 50 μM Mre11 inhibitor Mirin. Southern blot analysis was performed to analyze viral genome amplification of DNA digested with BamHI (nonviral genome cutter; upper panel) or HindIII (cuts viral genome once; lower panel). All results are representative of observations of two or more independent experiments.