Fig 2: The ECT2-Ex5+ splice variant promotes Doxo resistance and S phase accumulation. (A) Effects of siRNAs targeting either exon 5 (siECT2-ex5) or constitutive exons of ECT2 (siECT2tot) on MCF7-DoxoR cell survival in the absence and presence of Doxo 150 µM, as measured by WST1 assay and expressed as percent of control siRNA, as in Figure 2. (B) RT-qPCR analysis of ECT2 Ex5+ and Ex5- isoforms. (C–E) Cell cycle analysis by FACS in MCF7-DoxoR and MCF-7 cells treated for 48 hours with Doxo at 150 and 0.5 µM, respectively. DNA content was measured with propidium iodide (PI). Replicating DNA was pulse-labeled with BrdU either at the beginning (D) or at the end (E) of a 48-hour incubation with or without Doxo. (F) Effects of ZRANB2, SYF2 and ECT2-Ex5+ depletion on MCF7-DoxoR cell cycle in the absence and presence of Doxo. The proportion of cells in G1 and S phase was measured by FACS with PI staining.

Fig 3: Identification of exons regulated by ZRANB2. (A) Types of AS events regulated by ZRANB2 depletion for 48 h in MCF7-DoxoR cells (RNA-seq analysis). (B) Direction of regulation of ASE events by ZRANB2 depletion. (C) ZRANB2-regulated ASEs with a nearby ZRANB2-binding site found by CLIP-seq. In bold, events that were validated by RT-PCR for regulation by ZRANB2 (all the tested events were validated). (D) Enriched functions (Ingenuity Pathway Analysis) in genes with AS regulation by ZRANB2. (E) Comparison of ZRANB2-regulated AS events with AS events regulated in MCF7-DoxoR versus MCF-7 cells. (F) RT-PCR validations of AS events regulated by siRNAs targeting ZRANB2 (whose depletion was verified in Figure 2B) and in MCF7-DoxoR versus MCF-7 cells. The effects of ZRANB2 siRNAs on FGFR1OP were weak but reproducible.

Fig 4: ZRANB2 and SYF2 splicing programs converge on the ECT2-Ex5+ splice variant. (A) Overlap between AS events regulated by ZRANB2 and SYF2 in RNA-seq. Events that were regulated in the opposite direction in MCF7-DoxoR versus MCF-7 cells are underlined, and those that were validated by RT-PCR are in bold. (B) RT-PCR validation of AS regulation in the MAST2, MRPL55 and ECT2 genes and RT-qPCR analysis of ECT2 isoforms and total mRNA levels in MCF7-DoxoR versus MCF-7 cells, and in MCF7-DoxoR cells transfected with siRNAs targeting ZRANB2 and SYF2 (whose depletion was verified in Figure 2B and C). (C, D) RT-qPCR analysis of ECT2 isoforms and total mRNA levels (C), and WST1 analysis of Doxo survival (D, normalized to untreated cells as in Figure 2) in MCF7-DoxoR cells transfected with siRNA pools #1 targeting ZRANB2 (siZ) and SYF2 (siS) alone and in combination (siZ+S). ZRANB2 and SYF2 deletion are shown in Supplementary Figure S4F. (E) CLIP-qPCR analysis of ZRANB2 and SYF2 association with ECT2 pre-mRNA at the indicated locations. IgG, control immunoglobulin.