Fig. 6.

NEK2 modulates an RBFOX2-dependent pro-mesenchymal splicing program in TNBC cells. A Scatter plot of RNA expression levels of RBFOX2 and NEK2 across multiple ER⁻/HER2⁻ BC cell lines according to data of CCLE project. Pearson’s correlation coefficient (r) and associated p-value are shown. B qRT-PCR analysis of relative expression levels of RBFOX2 to L34 in control or NEK2-silenced MDA-MB-231 cells (mean ± SD, n = 3, *p-value≤0.05 t-test). C,D Western Blot (C) and densitometric analysis (D) of expression levels of RBFOX2, hnRNPL and NEK2 in si-CTRL or si-NEK2 MDA-MB-231 cells. HSP90 was evaluated as loading control (mean ± SD, n = 3, levels in si-CTRL were set to 1, *p-value≤0.05, ns = not significant, t-test). E) PCR and densitometric analysis of percentage of splicing inclusion (PSI) for indicated alternative splicing events in MDA-MB-231 cell transfected with control or NEK2, RBFOX2, hnRNPL targeting siRNAs (mean ± SD, n = 3, one-way ANOVA). Schematic representation for each event analysed is depicted beside relative agarose gels. Green and red boxes indicate down- and up-regulated exons in si-NEK2 vs si-CTRL cells. F,G Venn diagram showing alternative exon-cassettes overlapping (hypergeometric distribution test) in the comparison between control and NEK2-silenced cells and between epithelial and mesenchymal breast cancer cells from indicated study (F) and bar graph showing their regulation in the two comparison (Fisher’s exact test) (G). H PCR and densitometric analysis of PSI for indicated EMT-related cassette exons in HCC1937 and control or NEK2-silenced MDA-MB-231 cells (mean ± SD, n = 3, one-way ANOVA). Schematic representation for each event analysed is depicted beside relative agarose gels ad in (E). I Western Blot and densitometric analysis of the ratio between expression levels of NF-YA isoforms including or excluding exon 4 analysis for NF-YA in HCC1937 and control or NEK2-silenced MDA-MB-231 cells (mean ± SD, n = 4, one-way ANOVA). NEK2 and HSP90 were evaluated as control