Figure 2.

SOMCL-19-133 inhibits the NEDD8 pathway, which leads to DNA damage response, cell cycle arrest, and apoptosis. (a) Representative western blot showing the levels of UBA3^NEDD8, Ubc12^NEDD8, Cul^NEDD8 conjugates, and CRLs substrates in SOMCL-19-133 or MLN4924 treated cells. (b) SOMCL-19-133 induced a dose-dependent increase in DNA damage. HCT-116 and RKO cells were treated with various concentrations of SOMCL-19-133 or MLN4924 for 24 h. Induction of γH2AX, p-Chk1 (Ser317), and p-Chk2 (T68) was determined by western blot. (c and d) Effects of SOMCL-19-133 on cell cycle distribution and apoptosis. Cells were treated with SOMCL-19-133, MLN4924, or Control for (c) 24 h or (d) 48 h, and (c) DNA profiles were analyzed by PI staining-based flow cytometry; > 4N represents cells with greater than tetraploid DNA content. >4N cells were not included in the cell cycle distribution analysis. (d) Apoptotic cells were detected by Annexin V-FITC/PI staining-based flow cytometry. MLN represents MLN4924. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. (e) The levels of cleaved (Clvd) caspase-3, cleaved caspase-7, cleaved caspase-8, and cleaved PARP were assessed by western blot in cells treated with various doses (0, 0.1, 0.3, 1 µM) of SOMCL-19-133, MLN4924 for 48 h. (f) Western blot analysis of Noxa, Puma, BAD, BID, BAX, BAK, BCL-X_L, BCL-2, and MCL-1 in cells treated with SOMCL-19-133 or MLN4924 at 0, 0.1, 0.3, 1 µM for 48 h. All of the experiments were repeated three times.