Fig. 2

NEK2 regulates TNBC alternative splicing profile. A Western blot analysis for NEK2 and splicing factors SRSF1 and hnRNPF/H in cytosolic, nucleoplasmatic and chromatin extracts of BC cell lines. TUBULIN, MATR3 and H3 expression were evaluated as loading control of indicated fraction. B Representative images of confocal immunofluorescence analysis using an anti-NEK2 antibody in MCF-7 and MDA-MB-231 cells. DAPI staining was used to identify nuclear morphology. Insets show magnified images. Images were taken using a 60x objective lens. Scale bar 40 μm. C Venn diagram showing a significant overlap (hypergeometric distribution test) between genes regulated at the gene expression (GE) and alternative splicing (AS) level in the comparison between NEK2 silenced (si-NEK2) and control (si-CTRL) MDA-MB-231 cells. D Pie chart showing percentages of indicated different splicing pattern among regulated splicing events in the si-NEK2 vs si-CTRL comparison. E Representative PCR analysis for indicated alternative splicing events in si-CTRL or si-NEK2 MDA-MB-231 (left panel) and SUM159 (right panel) cells. 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. Percentage of splicing inclusion (PSI) of indicated exons was evaluated by densitometric analysis, and results are shown below agarose gels (mean ± SD, n = 3, t-test)