Figure 6.

Effect of CMLD011580 on multiple myeloma JJN3 cell viability. (a) Dose-dependent inhibition of protein synthesis by (−)-CR-1-31-b and CMLD011580 in JJN3 cells. Cells were incubated with the indicated compound concentrations for 1 h and [³⁵S]-methionine was added 15 minutes before the end of the incubation. The rate of [³⁵S]-methionine incorporation is expressed relative to that of cells treated with vehicle (DMSO). Results are expressed as mean ± SEM of 3 biological replicates. (b) Relative survival of JJN3 cells exposed to (−)-CR-1-31-b or CMLD011580 for 48 h with cell viability assessed by CellTiter Glo. Results are expressed as mean ± SEM of 3 biological replicates. (c) CMLD011580 induces apoptosis in JJN3 cells. Apoptosis was assessed 24 h after compound addition at the indicated concentrations by annexin V and propidium iodide staining. Presented is a representative image of 2 experiments. (d) Western blot of extracts prepared from JJN3 cells treated with the indicated compound concentrations (nM) for 5 h and probed with antibodies to the proteins indicated to the right. Blots were obtained from the same membrane sequentially probed with the indicated antibodies and are cropped for ease of comparison. (e) Polysome analysis of JJN3 cells treated with vehicle (0.05% DMSO) or 25 nM CMLD011580 for 1 h. Polysomes were fractionated on a 10–50% sucrose gradient. The distribution of MYC and GAPDH mRNA within the polysome fractions of vehicle (black boxes) or CMLD011580 (red boxes) treated cells was measured by RT-qPCR. Results are expressed as mean ± SEM of 2 biological replicates.