Fig. 3

By inhibiting neddylation, MLN4924 induces a robust UPR in mesothelioma cells. 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) for 24 h. In panel A and D, we showed the results of the MPM cells that resulted the top-responder cells to the cytotoxic effects of the combination of PT and MLN4924 (according to the results reported in Figure 2): epithelioid (EPI BAP1⁺ UPN7), biphasic (BIP BAP1^- UPN16) and sarcomatous (BAP1⁺ UPN22) MPM cells. A. Cullin 1 neddylation (NED, upper band) was measured by immunoblotting in. Tubulin was used as a loading control. The figure is representative of 1 out of 3 experiments with similar results on EPI BAP1⁺ UPN7, BIP BAP1^- UPN16 and SAR BAP1⁺ UPN22. B. UBE2M activity was measured spectrophotometrically in duplicates. 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. C. Proteasome activity was measured fluorometrically in duplicates. 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. D. Heatmap of the Unfolded Protein Response genes relative expression, in SAR BAP1⁺ UPN 22 MPM cells. Results are expressed in a logarithmic scale. The figure is representative of 1 out of 3 experiments with similar results. Cyan circles: EIF2AK3 and HSPA5 genes. E. EIF2AK3 and HSPA5 proteins were measured by immunoblotting in epithelioid (EPI BAP1⁺ UPN7), biphasic (BIP BAP1^- UPN16) and sarcomatous (BAP1⁺ UPN22) MPM cells. Tubulin was used as a loading control. The figure is representative of 1 out of 3 experiments with similar results