Fig. 2

Cisplatin+MLN4924 combination induces cell cycle alteration, DNA damage and senescence. 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 BAP^- UPN23) were incubated in fresh medium (CTRL), with 50 μM cisplatin (PT), 0.2 μM MLN4924 (MLN) or their combination (PT+MLN). A. Cell cycle distribution was measured by flow cytometry in duplicates, after 24 h (upper panels) or 48 h (lower panels). Data are presented as means of 2 primary MPM for each histopathological subtype used + SD (n = 3). *p<0.05, **p<0.01, ***p < 0.001: treated cells vs CTRL cells; °p<0.05, °°p < 0.01: PT+MLN-treated cells vs PT-treated cells. B. DNA damage was measured fluorometrically after 24 h. 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 < 0.05, °°°p < 0.001: PT+MLN-treated cells vs PT -treated cells. C. Left panel: representative sections of AB1 tumors from each group of animals, treated as indicated in Figure 1D-E, stained with the Ki67 antibody. Nuclei were counter-stained with hematoxylin. Scale bar: 100 μm (10x ocular; 40x objective). At least 10 fields were examined for each condition. Right panel: quantification of Ki67-positive cells versus the total number of cells, performed with the ImageJ software. Data are presented as means + SD. *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. D. Left panel: representative TUNEL staining on tumors sections from each group of animals, as above. Nuclei were counter-stained with DAPI. Scale bar: 200 μm. (10x ocular, 40x objective). At least 10 fields were examined for each condition. Right panel: quantification of TUNEL-positive cells versus the total number of cells, performed with the ImageJ software. Data are presented as means + SD. ***p < 0.001: treated cells vs CTRL cells; °°°p < 0.001: PT+MLN-treated cells vs PT-treated cells