Supplementary MaterialsAdditional file 1: Supplemental Figure 1. measurements were performed 72?h after knockdown. Supplemental Figure 3. Spliceosome candidate distribution over different subcomplexes(A) Candidate distribution over core and non-core complexes. (B) Candidate distributation of functional splicing subcomplexes. Supplemental Figure 4. Effect of candidate splicing factor knockdown on cell death and Bifendate cell cycle progression Bifendate in MDA-MB-231(A) Effect of splicing factor knockdown on cell DNA content measured by FACS analysis in MDA-MB-231 cells 72?h after transfection. Mean?+?stdev of three biological replicates. SRRT is a splicing factor not affecting proliferation and was used as negative control. (B) Effect of splicing factor knockdown on expression levels of cell cycle regulators in MDA-MB-231 cells 72?h after transfection. Representative blots of two biological replicates. SRRT Bifendate is a splicing factor not affecting proliferation and was used as negative control.Statistical significance was determined using ANOVA correcting for multiple testing. * Effect of SNRPD2, SNRPD3 and NHP2L1 knockdown in proliferation and cell death three days after knockdown in HCC1806 (A), MDA-MB-468 (B), MCF7 (C) and T47D (D) cells. Mean?+?stdev of 3 biological replicates. Significance was determined using ANOVA correcting for multiple testing. Supplemental Figure 6. SNRPD2, SNRPD3 and NHP2L1 knockdown reduce proliferation and induce cell death(A) Representative images of propidium iodide (PI) and Hoechst staining 2, 4 and 7?days after knockdown in MDA-MB-231 cells. Scale bar?=?100?m. (B) Cell number 2, 4 and 7?days after splicing factor knockdown in Hs578T (top) and MDA-MB-231 (bottom) cell lines. Cell growth was measured using the SRB absorbance. Mean?+?stdev of three biological replicates. Statistical significance was calculated using ANOVA correcting for multiple testing. * (A) Nuclear phenotype 72?h after ADFP splicing factor knockdown in MDA-MB-231 cells. Scale bar?=?50?m. (B) Effect of SNRPD2, SNRPD3 and NHP2L1 knockdown on nuclear phenotype in HCC1806, MDA-MB-468, MCF7 and T47D cells 72?h after transfection. Scale bar?=?20?m. Supplemental Figure 9. Effect of SNRPD2, SNRPD3 and NHP2L1 knockdown on nuclear phenotype and mitosis(A) Nuclear phenotype of p-histone H3 positive cells 72?h after SNRPD2, SNRPD3 or NHP2L1 knockdown in Hs578T. Some cells with irregular nuclear phenotype are p-histone H3 positive (white squares), while some are negative (blue squares). (B) Similar as A, but for MDA-MB-231. Scale bar?=?100?m. Supplemenatal Figure 10. Effect of candidate splicing factor knockdown on sister chromatid cohesion components(A) Overview of the factors involved in sister chromatid cohesion. Adapted from Peters et al., 2012. (B) Effect of 72?h SNRPD2, SNRPD3 and NHP2L1 knockdown on RNA expression levels of genes involved in sister chromatid cohesion in Hs578T. Mean?+?sd of 3 biological replicates.(C) Effect of 72?h SNRPD2, SNRPD3 and NHP2L1 knockdown on RNA expression levels of genes involved in sister chromatid cohesion in MDA-MB-231. Mean?+?sd of 3 biological replicates. (D) Effect of SNRPD2, SNRPD3 and NHP2L1 knockdown on sororin, ESPL1, MAU2 and SMC1 expression levels 1, 2 and 3?days after knockdown in MDA-MB-231. Mean?+?sd of 3 biological replicates. Statistical significance was determined using ANOVA correcting for multiple testing. * (A) Effect of 72?h SNRPD2, SNRPD3 and NHP2L1 knockdown on sororin intron 1 and 2 retention in Hs578T and MDA-MB-231 cells. Mean?+?sd of three biological replicates. (B) Effect of 72?h SNRPD2, Bifendate SRNPD3 and NHP2L1 knockdown on sororin intron 5 retention in Hs578T and MDA-MB-231 cells. Mean?+?sd of three biological replicates. (C) Effect of 72?h SNRPD2, SNRPD3 and NHP2L1 knockdown on sororin intron 1 and 2 retention 1, 2 and 3?days after knockdown in Hs578T and MDA-MB-231. Mean?+?sd of three biological replicates. Statistical significance was determined using ANOVA correcting for multiple testing. * Effect of SNRPD2, SNRPD3 and NHP2L1 knockdown on sororin intron 3 (A), intron 4 (B) or intron 5 (C) retention in Hs578T and MDA-MB-231 cell Bifendate lines 72?h after transfection. (D) Effect of candidate splicing factor knockdown on sororin intron 2 retention in MDA-MB-231 and Hs578T cells 72?h after transfection. SRRT is a splicing factor not affecting TNBC proliferation and was used as a negative.