Fig. 5

MLN4924-responsive cells have higher levels of SKP2. A-B. Primary mesothelioma cells of epithelioid (EPI; n= 10), biphasic (BIP; n = 5), sarcomatous (SAR; n =4) origin were incubated 72 h in fresh medium (CTRL), with 50 μM cisplatin (PT), 0.2 μM MLN4924 (MLN) or their combination (PT+MLN). Crystal violet staining was quantified spectrophotometrically. A: disaggregated data of cell viability. B: MPM cells divided in responders (samples where the combination of cisplatin+MLN4924 decreased the number of viable cells below 50%) and non-responders (samples where the combination of cisplatin+MLN4924 maintained >50% viable cell, red line). Data are presented as means + SD; each sample was analyzed in quadruplicates. * p < 0.05, **p < 0.01, ***p < 0.001: treated cells vs CTRL cells; °°°p < 0.001: PT+MLN-treated cells vs PT -treated cells. C-F. Top responder (the epithelioid EPI BAP1⁺ MPM UPN7, the biphasic BIP BAP1^- MPM UPN16, the sarcomatous SAR BAP1⁺ MPM UPN22) and top non-responder (the epithelioid EPI BAP1⁺ MPM UPN6, the biphasic BIP BAP1⁺ MPM UPN18, the sarcomatous SAR BAP1^-MPM UPN21) cells were incubated as in A-B for 24 h C. SKP2 mRNA levels were measured by RT-PCR, in triplicates. Data are presented as means + SD (n = 3). ***p < 0.001: treated cells vs CTRL cells, °°°p < 0.001: treated cells vs PT-treated cells; ^###p < 0.001: non-responder cells vs respective responder cells. D. The expression of SCF complex proteins – cullin 1, SKP1 and SKP2 - was measured by immunoblotting. Tubulin was used as a loading control. The figure is representative of 1 out 3 experiments with similar results. E. UBE2M activity was measured spectrophotometrically, in duplicates. Data are presented as means + SD (n = 3). ***p < 0.001: treated cells vs CTRL cells, °°°p < 0.001: treated cells vs PT-treated cells; ^###p < 0.001: non-responder cells vs respective responder cells. F. Proteasome activity was measured fluorometrically, in duplicates. Data are presented as means + SD (n = 3). ***p < 0.001: treated cells vs CTRL cells; °°p < 0.01: treated cells vs PT-treated cells