14-3-3ε modulation affects sensitivity to PIs in MM cells in vitro and in vivo. (A) Control and ectopically expressing 14-3-3ε KMS20 cells were treated with different concentrations of BTZ or CFZ. Cell viability was assessed by CTG after 24 hours of treatment and expressed as percentage change from untreated cells (top). IC₅₀ analysis is also shown (bottom). Data represent mean ± SD from 3 experiments performed in triplicate. (B) Control and ectopically expressing 14-3-3ε LR5, KMS26, and SKMM1 cells were treated with different concentrations of BTZ or CFZ. IC₅₀ is shown in the graph. Data represent mean ± SD from 3 experiments performed in triplicate. (C) YWHAE was knocked out in KMS11 with the CRISPR/Cas9 system, and then a single clone of KO cells was infected with either pLenti6-empty (empty) or pLenti6-FLAG-YWHAE OE plasmid (14-3-3ε). Expression of 14-3-3ε protein was evaluated by western blot. GAPDH was used as loading control (right). Infected cells were treated with different concentrations of BTZ or CFZ. Cell viability was assessed by CTG after 24 hours of treatment, and IC₅₀ was calculated with GraphPad Prism 8 (left). (D) Control and ectopically expressing 14-3-3ε ANBL-6 WT and ANBL-6/V10R cells were treated with different concentrations of BTZ or CFZ. IC₅₀ analysis is shown. Data represent mean ± SD from 3 experiments performed in triplicate. (E) Control and ectopically expressing 14-3-3ε KMS20 cells were injected subcutaneously in severe combined immunodeficiency mice. After detection of tumor, mice were treated either with placebo or 2 cycles of BTZ (0.5 mg/kg twice weekly for 2 weeks, followed by 2 weeks off). Tumor growth was evaluated weekly by caliper measurement and represented as tumor volume (millimeters cubed). (F) Comparison of tumor volume in control and treated mice after the first (left) and second (right) cycle of treatment, respectively. Data were analyzed using unpaired Student t tests: *P ≤ .05; **P ≤ .01; ***P ≤ .001; ****P ≤ .0001.