(A) SF3A2 and PRP31 localization in HeLa cells; both SFs are slightly enriched in the central part of the spindle region; in nocodazole (Noc)-treated cells the SFs are diffuse in the mitotic cytoplasm; in SF3A2 or PRP31 RNAi cells, the spindles are not immunostained by the pertinent antibodies. In merged images, DNA is blue, tubulin green and the SFs red. Scale bar, 5 μm. (B) Western blotting showing that RNAi-mediated depletion of SF3A2 or PRP31 strongly reduces the levels of these proteins without affecting HEC1 expression. Actin is a loading control. Quantification of the HEC1 level from three independent experiments does not reveal significant differences between control and RNAi cells using unpaired t-test. Error bars, SEM. (C) Mitotic index (M.I.) and frequencies of mitotic figures observed after RNAi against SF3A2 and PRP31. R, are specific rescue constructs expressing RNAi resistant SF3A2 and PRP31 genes. (D) Representative prometaphase figures (not treated with nocodazole) stained for DNA, HEC1 and tubulin showing that RNAi against SF3A2 or PRP31 results in the formation of a HEC1 halo surrounding the chromosomes. The graph shows quantification of halo fluorescence. To estimate the fluorescence intensities of the halos, we calculated the signal-to-background ratios in 20 randomly chosen cells, as described in Materials and methods. In SF3A2 and PRP31 RNAi cells, these ratios are significantly higher than that detected in control cells (p<0.001 for both samples in the unpaired t test).(E) Representative nocodazole-treated mitotic figures stained for tubulin and HEC1. The graph shows a quantification of the HEC1 fluorescence associated with the chromosomes; see Materials and methods for details on fluorescence measurement. The fluorescence intensities detected in SF3A2 and PRP31 RNAi cells are not significantly different from that of control cells in the unpaired t test.