Figure 2.

MLN4924 Inhibits Ku Removal from DNA Repair Sites

(A and B) MLN4924 causes Ku80 foci persistence after IR. U2OS cells were pre-treated with DMSO or 3 μM MLN4924 for 1 hr and then subjected to 10 Gy IR. Samples were pre-extracted with CSK+RNase A and visualized by immunofluorescence. (A) shows representative images, and (B) shows quantification. Dotted lines indicate nuclear peripheries. Error bars correspond to SDs of at least three independent experiments (asterisks as in Figure 1B). White bar represents 10 μM.

(C) U2OS-A171T UBA3 are resistant to MLN4924. U2OS cells stably expressing WT UBA3 or A171T UBA3 were treated with DMSO or 3 μM MLN4924 for 1 hr and analyzed by immunoblotting with indicated antibodies. Endogenous UBA3 was depleted with a siRNA to the 3′ UTR (Figure S2B). Neddylated conjugates are detected with a NEDD8-specific antibody.

(D) MLN4924 effects on Ku80 foci retention are through UBA3 inhibition. U2OS cells stably expressing WT UBA3 or A171T UBA3 were processed as in (A), and results were quantified as in (B).

(E) MLN4924 does not affect γH2AX recovery after IR. Quantification of total γH2AX intensity per nucleus in cells treated with 10 Gy IR then harvested at indicated times. Samples were prepared as in (A). Pre-treatment for 1 hr with 3 μM DNA-PK inhibitor (DNA-PKi) used as a positive control. Statistical analysis as in (B). AU, arbitrary units. See also Figure S2.