Fig. 6.

SKP2 levels controls the sensitivity to cisplatin and MLN4924 in malignant pleural mesothelioma cells. Top responder cells (EPI BAP1⁺ MPM UPN7) to the combination cisplatin+MLN4924 were transduced with a non-targeting scrambled vector (SCR) or with a CRISPR/Cas9 SKP2-knocking out vector (KO). Top non-responder cells (EPI BAP1⁺ MPM UPN6) were transduced with an empty vector (EM) or with an expression vector for SKP2 (OVER). A. SKP2 expression was measured by immunoblotting. Tubulin was used as a loading control. The figure is representative of 1 out of 3 experiments with similar results. B-D. Cells were incubated for 48 h (panel B) or 72 h (panel C-D) in fresh medium (CTRL) with 50 μM cisplatin (PT), 0.2 μM MLN4924 (MLN) or their combination (PT+MLN). B. LDH release, taken as cytotoxicity index, was measured spectrophotometrically in triplicates. Data are presented as means + SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001: treated cells vs CTRL cells; °°p < 0.01, °°°p < 0.001: PT+MLN-treated cells vs PT -treated cells. C. Representative photographs of crystal violet staining, from 1 of 3 experiments. D. Crystal violet staining was quantified spectrophotometrically. Data are presented as means + SD; each sample was analyzed with technical quadruplicates. ***p < 0.001: treated cells vs CTRL cells; °°°p < 0.001: PT+MLN-treated cells vs PT -treated cells. E. SKP2 expression was measured by RT-PCR in samples of each patient (n=19) receiving platinum-derivatives as first-line treatment, and median value was calculated. Patients were classified as SKP2^low (blue line, including 10 patients) and SKP2^high (green line, including 9 patients) if the mRNA levels were low or equal/higher than the median value. Time to progression (TTP; left panel) and overall survival (OS; right panel) probability was calculated using the Kaplan-Meier method. *p<0.016 (TTP); *p<0.044 (OS): SKP2^high vs SKP2^low group