Fig. 6: In vitro experiments following modulation of key splicing factors: overexpression of HNRNPK and MBNL1 (1 µg/mL each) and silencing of PTBP1 (75 nM).

A Validation of each modulation at the RNA level by qPCR. The graphs show the expression levels of each splicing factor relative to the control (CMV6 plasmid for overexpression; scramble for silencing) 24 h post-transfection. Expression was normalized using GAPDH as the housekeeping gene. The dotted line at Y = 100 represents the control. B Cell viability assay after each transfection. Cell viability rate is shown as percentage of the control (dotted line at Y = 100) at the different times (24, 48, 72 h). C Cancer-related genes altered after modulation of HNRNPK, MBNL1, and PTBP1, measured by qPCR. Gene expression is presented as Log₂ of the Fold-Change modulation/control. Upregulated genes are shown in red, downregulated genes in blue. D RNA expression level of mucin 2 splicing variants after HNRNPK, MBNL1 and PTBP1 modulation. Expression levels of MUC2 isoforms (MUC2-204, MUC2-205, and MUC2-206) are shown in the control transfection (CMV6 plasmid for HNRNPK and MBNL1, or scramble for PTBP1), normalized to GAPDH expression. Data are presented as mean ± SEM, n = 3. For cell viability, differences were assessed using ANOVA with post hoc Tukey’s test for pairwise comparisons. For other 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. E Relative expression of MUC2-205 and MUC2-206 splice variants in N14A after modulation of HNRNPK, MBNL1, and PTBP1. PSI (percent spliced-in) for MUC2-205 and MUC2-206 splice variants are shown for control and different modulations.