Fig. 2

Effect of candidate splicing factor knockdown on cell death and cell cycle progression. a Results of validation screen; effect of SMARTpool and 4 single siRNAs of selected candidates on proliferation (SRB and nuclei count) and cell death (Annexin V and PI staining) in Hs578T cells 72 h after knockdown. PPIH, SRPK2 and SRRT were splicing factors not affecting proliferation in the primary screen and used as negative control. b Effect of splicing factor knockdown on cell DNA content in Hs578T cells measured by FACS analysis 72 h after knockdown. Mean + stdev of three biological replicates. siSRRT was used as a splicing factor negative control. c Effect of selected splicing factor knockdown on cell cycle arrest measured using the FUCCI cell cycle system in Hs578T cells 72 h after knockdown. Mean + stdev of three biological replicates. siSRRT was used as a splicing factor negative control. d Example of the effect of splicing factor SNRPD3 knockdown on FUCCI cell cycle markers in Hs578T cells. e Effect of splicing factor knockdown on expression levels of cell cycle regulators in Hs578T cells. Representative blots of two biological replicates. f Representative images of propidium iodide (PI) and Hoechst staining 2, 4 and 7 days after knockdown in Hs578T cells. Scale bar = 100 μm. g Percentage of cell death 2, 4 and 7 days after splicing factor knockdown in Hs578T (top) and MDA-MB-231 (bottom) cell lines. Mean + stdev of three biological replicates. h Effect of splicing factor knockdown on caspase activity in Hs578T (top) and MDA-MB-231 (bottom) cell lines 4 days after transfection. Mean + stdev of three biological replicates. Statistical significance was determined using ANOVA correcting for multiple testing. * p < 0.05, ** p < 0.01, *** p < 0.001