Fig 2: Global effects of SF3B1 on AS based on RNA-seq. (A) Reduced expression of SF3B1 in HCT116 cells treated with an SF3B1-targeting shRNA or a non-silencing shRNA based on RT-PCR and immunoblotting using ACTB for RT-PCR and a-tubulin and U2AF65 for immunoblotting controls. (B) Pie chart of RNA-seq results showing alteration quantities of SEs, A5SS, A3SS, MXE, and RI in SF3B1 KD cells. (C) Pie chart of gene distributions of altered SEs in SF3B1 KD cells. (D–H) Gene ontology analysis of AS-regulated genes in SEs, A5SS, A3SS, MXE, and RI of SF3B1 KD cells.

Fig 3: Validation of regulated A5SS, A3SS, and RI in SF3B1 KD cells. (A–C) (Left) Schematic representation of alternative splice sites in genes. Sequences of two alternative 5'SSs (A); two alternative 3'SSs and PPTs (B); and 5'SSs, 3'SSs, and PPTs of introns (C) are shown. (Middle) Sashimi plots of validated A5SS (A), A3SS (B), and RI (C) in RNA-seq. (Right) RT-PCR analysis of altered A5SS (A), A3SS (B), and RI (C).

Fig 4: SF3B1 is required for an efficient DDR. A, Clonogenic survival assays demonstrating that depletion of SF3B1 (two independent siRNAs) induces sensitivity to IR in 293T cells. Mean surviving fraction of three independent experiments is plotted ± SEM. B, Representative immunofluorescence staining of ?H2AX-marked DNA damage in untreated U2OS cells depleted of SF3B1, and 1 and 24 hours following 2 Gy IR. White scale bar, 20 µm. C, Quantification of three independent experiments described above (=200 cells were scored/experiment). Mean percent cells containing =5 ?H2AX foci is plotted ± SEM. D, Quantification of GFP-positive cells by flow cytometry from a DR-GFP reporter cassette stably integrated in U2OS cells, to assess HR. Mean % GFP-positive cells normalized to siSCR-transfected cells is plotted ± SEM. E and F, Long-term cellular survival assays demonstrating that depletion of SF3B1 (two independent siRNAs) induces sensitivity to etoposide and olaparib in U2OS cells and that SF3B1 and BRCA1 are epistatic. Mean surviving fraction of three independent experiments is plotted ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; NS, nonsignificant.

Fig 5: SF3B1 binds to PPT7 more strongly than to PPT8. (A) Sequences of biotin-labeled PPT7 and PPT8 oligonucleotides are shown. (B) RNA-IP with the anti-SF3B1 antibody using PPT7 and PPT8 oligonucleotides in HEK293T cell lysates. (C) UV crosslinking/IP with the anti-SF3B1 antibody using PPT7 and PPT8 oligonucleotides in HEK293T cell lysates.