Flux and turnover assays for evaluating Abx-induced autophagic response in myeloma cells. (A) Change in LC3-II expression levels in myeloma cells by Abx treatment (flux assay). Western blots revealed time course increases in LC3-II expression in KMS21 and KMS34 cells after 100 μM Abx treatment. ∗P < .05 vs 0 hour. (B) Change in p62/SQSTM1 expression in myeloma cells by Abx treatment. Time course changes in p62/SQSTM expression were evaluated by western blots in KMS21 and KMS34 cells after treatment with 100 μM Abx. ∗P < .05 vs 0 hour. (C) Turnover assay of myeloma cells by Abx. KMS21 and KMS34 cells were exposed to 100 μM Abx for 24 hours and cotreated with 100 μM resveratrol (Res) for an additional 18 hours or 10 nM bafilomycin (Baf) A1 for an additional 2 hours. Changes in LC3-II expression were evaluated by western blot. (D) Reporter assay for evaluation of LC3-II turnover. pMRX-IP-GFP-LC3-RFP-LC3ΔG–transduced KMS34 cells were treated with 0, 200, or 400 μM Abx for 48 hours. Cells were cocultured with 100 μM Res or 50 nM Baf A1 for an additional 12 hours. The effect on autophagy was evaluated by fluorescence microscopy. GFP levels were semiquantified by flow cytometry using FlowJo software. Ax, ambroxol; ctrl, control.