JS-K induces DNA DSBs, and activates DNA damage response pathways. (A) MM.1S cells were treated with JS-K (0-2.5 μM) for 2.5 hours, and then assayed for formation of DSBs by the neutral comet assay. Representative images of the comet tails are shown (left panel), and the olive tail moments are plotted (right panel). To calculate the olive tail moments, at least 40 cells per sample were analyzed. Data represents the mean (± SD) of 3 independent experiments. *P < .05 compared with nontreated control. (B-C) MM.1S cells were dosed with 2.5 μM JS-K for 0 to 4 hours. Representative images for the immunocytochemistry assay performed with antiphospho(Ser139)-H2AX antibody is shown (B). Phosphorylation of the DNA damage response proteins H2AX (Ser139), Chk1 (Ser317), Chk2 (Thr68), and p53 (Ser20) was assessed by Western blotting (C). Representative western blots (left panel), and percentage of mean quantitative densitometric values (± SD) from 2 or 3 independent experiments (right panel) are shown. *P < .05; **P < .01 compared with 0-hour control. (D) MM.1S cells were cultured with JS-K (0 to 2.5 μM) for 2 hours, after 1 hour of preincubation with cobalamin (50 μM). Phosphorylation of H2AX was assayed by Western blotting. Representative western blots and percentage of mean quantitative densitometric values ± SD from 2 independent experiments are shown. *P < .01 compared with no-cobalamin control. (E) Low doses of bortezomib sensitize MM cells to JS-K. MM.1S cells were treated with bortezomib (0, 1, 2, or 3 nM) for 8 hours, which was followed by JS-K treatment (0, 0.3, or 0.6 μM). Cell viability was detected by MTT assay (48 hours). Data represents the mean (± SD) of 3 independent experiments.