Flux assays and turnover assays of the effects of antimyeloma drugs used singly or in combination with Abx. (A) Change in LC3-II and p62/SQSTM1 expression levels in myeloma cells by existing antimyeloma drugs (flux assay). KMS34 cells were incubated with various concentrations of bortezomib (Btz), lenalidomide (Len), and panobinostat (Pan) for 72 hours, and the protein expressions were evaluated by western blots. ∗P < .05 vs 0 nM. (B) Turnover assay using existing antimyeloma drugs. For Btz, KMS34 cells were incubated with 8 nM Btz for 3 hours, after which 1 nM Baf A1 or 100 μM Res was added. The cells were then incubated for an additional 3 and 12 hours, respectively. For Len, Len-sensitive MUM24 cells were cultured with 1 μM Len for 48 hours. Then, 1 nM Baf A1 or 100 μM Res was added to Len-pretreated MUM24 cells, and the cells were incubated for an additional 3 or 12 hours, respectively. For Pan, KMS34 cells were treated with 20 nM Pan for 48 hours. Then, 1 nM Baf A1 or 100 μM Res was added to Pan-pretreated KMS34 cells, and the cells were incubated for an additional 3 or 12 hours, respectively. LC3B-II and p62 levels were evaluated by western blots. ∗P < .05. (C) Synergism of Abx with known antimyeloma drugs. KMS21, KMS34, and MUM24 cells were incubated with various concentrations of Abx plus Btz, Len, or Pan for 72 hours. Cell viability was evaluated by WST-1 assay. CIs were calculated by the Chou-Talalay method. Fa-CI plots are illustrated.14 (D) Growth inhibition by combination treatment of Abx plus Pan with or without Btz was evaluated by WST-1 assay. KMS21 and KMS34 cells were incubated with various concentrations of Abx in the presence of Pan and Btz for 72 hours. Abx0.5 (0.5 viability) compared with no drug treatment (1.0 viability) of the KMS21 and KMS34 cells are also found as Abx0.5. (E) MCL1 expression by Abx with antimyeloma drugs. KMS21 and KMS34 cells were treated with 200 μM Abx plus 8 nM Btz or 20 nM Pan for 24 hours. MCL1 expression was evaluated by western blot. ∗P < .05; ∗∗P < .01. (F) Western blot indicating caspase activation by combination treatment of Abx with Pan or Btz. KMS21 cells were treated with 200 μM Abx and 20 nM Pan or 8 nM Btz for 24 hours unless otherwise stated. (G) Expression levels of sirtuin 2, FOXK1, and ATG products and the level of UPR by combination treatment with 200 μM Abx and 20 nM Pan for 24 hours in KMS34 cells. RNA sequence analysis revealed that the Sirtuin 2 and FOXK1 gene expressions were upregulated by Abx treatment (Figure 7A-B). ctrl, control; Fa, fractional effect.
