Differential sensitivity to proteasome stress in MMCs. (A) Basal and PI-induced accumulation of polyubiquitinated proteins in MMCs. Immunofluorescent staining of polyubiquitinated proteins in U266, RPMI8226, KMS.18, and MM.1S cells. Top panels show untreated cells. Bottom panels show cells treated with bortezomib (Btz) for 24 hours at the corresponding EC₅₀ dose (calculated at 48 hours). One of 3 independent experiments is shown. Size bar equals 10 μm. (B) Accumulation of polyubiquitinated proteins and lower levels of free ubiquitin in PI-sensitive MMCs. Extracts from U266 and MM.1S cells were blotted with antiubiquitin (Ub). Polyubiquitinated proteins are detected as a smear in a 10% gel, while free Ub is detected in an 18% gel. β-actin serves as a loading control. (C) MM.1S and U266 were engineered to stably express Ub^G76V-GFP, an established in vivo reporter of proteasomal overload.^13,23,29  FACS analysis of basal Ub^G76V-GFP expression and its accumulation upon 4 hours of treatment with Btz (1 μM for U266 and 100 nM for MM.1S). (D,E) Ub^G76V-GFP accumulation upon 24 hours of treatment reveals critical proteotoxicity levels. Ub^G76V-GFP–engineered U266 and MM.1S were treated with increasing doses of Btz for up to 48 hours. (D) FACS analysis of GFP expression reveals massive accumulation of GFP upon 24 hours of treatment in both MM.1S and U266 with 5 and 50 nM Btz, respectively. (E) Mean fluorescence intensity (MFI) at different doses reveals a critical accumulation of the reporter at 24 hours (left panel), 1 day before onset of apotosis (right panel).