Fig. 1

Cisplatin and MLN4924 combination induce mesothelioma cell death. Primary MPM cells derived from 3 different histopathological subtypes, epithelioid (EPI BAP1⁺ UPN7 and EPI BAP1^- UPN11), biphasic (BIP BAP1⁺ UPN14 and BIP BAP1^- UPN16) and sarcomatous (SAR BAP1⁺ UPN22 and SAR BAP1^- UPN23), were incubated 48 h in fresh medium (CTRL), with 50 μM cisplatin (PT), 0.2 μM MLN4924 (MLN) or their combination (PT+MLN). A. LDH release was measured spectrophotometrically in triplicates. Data are presented as means of 2 primary MPM for each histopathological subtype used + SD (n = 3). ***p < 0.001: treated cells vs CTRL cells; °°°p < 0.001: PT+MLN-treated cells vs PT-treated cells. B. Representative crystal violet staining of epithelioid (EPI; UPN7), biphasic (BIP; UPN16) and sarcomatous (SAR; UPN22) MPM cells. The photographs are representative of 1 out of 3 experiments, performed in quadruplicates. C. Crystal violet quantification after incubation for 72 h in fresh medium (CTRL), with 50 μM cisplatin (PT), 0.2 μM MLN4924 (MLN) or their combination (PT+MLN) of epithelioid (EPI BAP1⁺ UPN7 and EPI BAP1^- UPN11), biphasic (BIP BAP1⁺ UPN14 and BIP BAP1^- UPN16) and sarcomatous (SAR BAP1⁺ UPN22 and SAR BAP^- UPN23) MPM cells. Data are presented as means of 2 primary MPM for each histopathological subtype used + SD (n = 3), performed in quadruplicates. ***p < 0.001: treated cells vs CTRL cells; °°°p < 0.001: PT+MLN-treated cells vs PT -treated cells. D. AB1 cells were subcutaneously implanted into 6-week-old female BALB/c mice, when the tumor size reached 50 mm³ mice (n = 10 animals/group) were treated for 3 consecutive weeks as indicated in the Methods section. Upper panel: Tumor growth was monitored twice a week by caliber measurement. Data are presented as means ± SD. *p < 0.05, **p < 0.01: all groups vs CTRL group; °p < 0.05: PT+MLN group vs PT group. Lower panel: Representative photographs of tumors from each group. E. Epithelioid (EPI BAP1⁺ UPN7), biphasic (BIP BAP1⁺ UPN14) and sarcomatous (SAR BAP1⁺ UPN22) cells, i.e. one MPM representative per each histotype, were incubated with scalar concentrations (0, 10^-10, 10^-9, 10^-8, 10^-7, 10^-6, 10^-5, 10^-4, 10^-3 M) of MLN4924 and cisplatin for 72 h, then the viability was assessed by the crystal violet staining. The Combination Index (CI) was calculated using the CalcuSyn software. Results are representative of 3 independent experiments, performed in quadruplicates. ED50: dose that effectively reduces cell viability at 50% with single agent or their combination