Fig. 5: In vitro experiments inhibiting splicing process using Pladienolide-B in the PMP cell line model N14A.
A Cell viability assay using Pladienolide-B at 10-10 M, 10-9 M, and 10-8 M. The graph shows cell viability percentage respect to the control of N14A in crescent doses of Pladienolide-B at the different times of the experiment (24, 48 and 72 h). The dotted line at Y = 100 represents the control (vehicle). B Cell viability assay using Pladienolide-B (10-9 M) in combination with mitomycin-C (4 µg/mL) and cisplatin (0.5 × 10-3 M). The graph shows the percentage of cell viability relative to the control (vehicle, dotted line at Y = 100) in N14A cells under different treatments: Pladienolide-B, mitomycin-C, cisplatin, Pladienolide-B + mitomycin-C, and Pladienolide-B + cisplatin at 24, 48, and 72 h. C Cell migration using Pladienolide-B at 10-9 M in the wound-healing assay. The left panel represents the quantification of the area of the scratch in the cells treated respect to the control in percentage in N14A cell line after 16 h. The dotted line at Y = 100 represents control. In the right panel are the pictures of the scratch at 16 h for the control (up) and the treatment (down). D Turbidity assay using Pladienolide-B at 10-9 M. OD at 900 nm is represented for the control (white box) and treated (colored box) cells. E RNA expression level of mucin 2 splicing variants using Pladienolide-B at 10-9 M. The RNA expression levels of all MUC2 isoforms, MUC2-204, MUC2-205 and MUC2-206 are represented in the control and treated cells. Data are represented as mean ± SEM, n = 3 in viability, migration and qPCR assays and n = 6 in turbidity assay. For cell viability, mean differences were assessed using ANOVA, with post hoc Tukey’s test to compare between group pairs. In the rest of the cases, t-test or Mann–Whitney U test were used. Asterisks represent significant differences compared to the control: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; symbol plus represents significant differences between the different treatments: Pladienolide-B vs Pladienolide-B + mitomycin-C; Pladienolide-B vs Pladienolide-B + cisplatin; mitomycin-C vs Pladienolide-B + mitomycin-C; cisplatin vs Pladienolide-B + cisplatin; +p < 0.05; ++p < 0.01; ++++p < 0.0001. F MUC-205 and MUC-206 splice variants relative expression in N14A. PSI (percent spliced-in) is represented for MUC-205 and MUC-206 splice variants in the control and after treatment (Pladienolide-B at 10-9 M). The structure of each mucin 2 variant is represented in the right scheme. Pd: Pladienolide-B.
