Fig. 2

CELF2 (CUGBP and ETR-3-like factor 2) tumor growth-inhibitory properties and its impact in alternative splicing patterns. a Efficient recovery of CELF2 expression upon transduction in MCF7 cells according to quantitative real-time-PCR (qRT-PCR) (left) and western blot (right). Triplicate qRT-PCR values were analyzed and expressed as the mean ± SD. b The colony formation assay showed that MCF7 cells stably transduced with CELF2 formed significantly fewer colonies than empty vector-transduced cells. Data shown represent mean ± SD of biological triplicates. P values are those corresponding to Student’s t test. c Empty vector (EV) and CELF2-transduced MCF7 cells were injected in the mammary fat pad of nude mice to form orthotopic tumors. Tumor volume over time (left) and tumor weight upon sacrifice (right) are shown. P values are those corresponding to Student’s t test. Bars show means ± SD. d Workflow of the RNA-sequencing (RNA-seq) analysis developed to detect RNA alternative splicing changes in MCF7 cells upon CELF2 transduction. e Gene ontology analysis (GO) of the RNAs with differential splicing in MCF7 cells upon CELF2 transduction (hypergeometric test with a false discovery rate (FDR) adjusted p value < 0.05). f Validation of CELF2 splicing targets in breast cancer cells. Restoration of CELF2 expression by transduction in CELF2 hypermethylated MCF7 cells induces a shift in the splicing patterns of NPTN, ULK1, CARD10, RHBDF2, and FBXL2, determined by exon-specific qRT-PCR. P values are those corresponding to Student’s t test. Bars show means ± SD. *p < 0.05, **p < 0.01, and ***p < 0.001