Figure 5. Role of MSLN in cellular growth, cell cycle progression and apoptosis, following treatment with chemotherapeutic drugs.

A. Proliferation assay in Mero-14 cells. The graph shows the effect of the treatments with 5 µM cisplatin and 40 nM siMSLN-1, used as single agents or in combination. On day 6, MANOVA shows a statistically significant effect both for cisplatin (P = 0.0168) and siMSLN-1 (P<10⁻⁴) in reducing proliferation. However, the interaction term for the effect of both agents in combination is not statistically significant (P = 0.145). Error bars represent SEM of three independent experiments, each performed in quadruplicate. B. Flow cytometry analysis. The graph shows the percentage of cells in phase S+G2+M in Mero-14 cells treated with 40 nM of the siCtrl or siMSLN-1 in combination with imatinib (25 µM) or gemcitabine (1 µM) (alone) or imatinib+gemcitabine (10 µM and 1 µM, respectively). The transfection with siMSLN-1 was accompanied with a marked decrease of cells in S+G2+M phase, as compared with the respective cultures transfected with siCtrl, irrespectively of the drugs employed (P =  0.00033). Error bars represent SEM of two independent experiments. C. Caspase activity measured on Mero-14 cells transfected with 40 nM of siCtrl, or siMSLN-1, with or without cisplatin 5 µM. A marked increase in apoptosis is observed when siMSLN-1 and cisplatin are administered together, compared to cultures treated with cisplatin and transfected with siCtrl (*P = 0.018), suggesting a synergistic effect. Error bars represent SEM of three independent experiments, each performed in triplicate. D. Western blotting analysis of MSLN, p53, and PARP under different combinations of siRNAs and cisplatin (at 5, 10 and 20 µM). β-actin was used as reference. The protein levels were confirmed with three independent experiments. Legend to figure 5: Dark line: cells trated with siCtrl; gray line and triangles: cells treated with siCtrl plus cisplatin; gray line and dark spots: cells treated with siMSLN plus cisplatin; dark line and white spots: cells treated with siMSLN-1.