Fig. 4

Alternative cassette exons in EZH2 and MDM4 genes affects 22Rv1 cells viability. A, B Kaplan Meier curves illustrating the progression (A) and disease free interval (B) of prostate adenocarcinoma (PC) patients of the TCGA cohort, segregated according to median expression levels of the EZH2 gene. C Schematic representation and profile of the RNA-seq reads of the indicated region of EZH2 gene in 22Rv1 cells treated with Indisulam (Ind) or Dmso. Sequence reads (vertical gray lines), exons (blue boxes), and introns (horizontal lines) are shown. Dashed red box highlights the RNA-seq reads along the upregulated exon 12 (red box). D Schematic representation of EZH2 exon 12 alternative cassette exon. Black arrows indicate primers used for the RT-PCR analyses of its inclusion in 22Rv1 treated or not with Ind (3,3 µM, 12 h). Percentage of spicing inclusion (PSI) is shown below the representative agarose gel (densitometric analysis, mean ± SD, n = 3, one-way Anova). NMD, non-sense mediated decay. E Representative Western Blot analysis for EZH2 protein levels in 22Rv1 cells treated or not for 24 h with Ind (3,3 µM). ACTIN was evaluated as loading control. Densitometric analysis of the EZH2/ACTIN ratio is shown below the blots (mean ± SD, n = 4, t-test). F Line graph showing growth rate of 22Rv1 cells treated with the EZH2 inhibitor GSK343 (10 µM) and Indisulam (3,3 µM), evaluated as cell confluence ratio relative to time 0 (t0; mean ± SD, n = 4, two-way Anova). G, H) Kaplan Meier curves illustrating the progression (G) and disease (H) free interval of PC patients of the TCGA cohort, segregated according to median expression levels of the MDM4 gene. I Schematic representation and profile of the RNA-seq reads of the indicated region of MDM4 gene in 22Rv1 cells treated with Pladienolide B (PladB) or Dmso. Sequence reads (vertical gray lines), exons (blue boxes), and introns (horizontal lines) are shown. Dashed green box highlights the RNA-seq reads along the downregulated exons 7 and 10 (green boxes). J Schematic representation of the alternative cassette exon events involving exon 7 and 10 of MDM4 gene. Black arrows indicate primers used for the RT-PCR analyses. K, L RT-PCR analysis of MDM4 exons 7 and 10 inclusion (upper panels) and western blot analysis (lower panels) of MDM4 protein levels in 22Rv1 treated or not with Plad B (10 nM, 6 h) (K) and in 22Rv1 transduced with control or MDM4 exon 7 targeting antisense oligonucleotide (ASO). ACTIN was evaluated as loading control. Results of the densitometric analysis of the PSI are shown below the agarose gel (mean ± SD, n = 3, t-test). Densitometric analysis of the MDM4/ACTIN ratio is shown below the blots (mean ± SD, n = 3, t-test). M) Line graph showing growth rate of 22Rv1 cells transduced with ASO-CTRL or ASO-MDM4 or treated with PladB (1 nM) or Dmso, evaluated as cell confluence ratio relative to t0 (mean ± SD, n = 2, two-way Anova)