TOE1 is required for maintaining Cajal body integrity and efficient splicing. (A) TOE1 was down regulated in HeLa cells transfected by siRNA against TOE1. (B) Knockdown of TOE1 affected the number and homogeneity of coilin foci. A control and two different siRNAs against TOE1 were used to knock down endogenous TOE1 expression in HeLa cells. Localization of TOE1 and coilin was detected by immunostaining using anti-TOE1 and anti-coilin. To recover the expression of TOE1, a construct encoding an siRNA-resistant form of TOE1 was cotransfected with siTOE1-A. The exogenous protein was detected by the anti-Flag antibody. (C) The bar graph shows the percentage of cells (±SD) containing more than four coilin foci after the indicated treatment. WT, wild type. (D and E) Down-regulation of TOE1 disrupted localization of SMN complex and newly synthesized Sm-D1. Control siRNA (siCTL)– or siTOE1-A–treated cells were subjected to coimmunostaining using anti-coilin and anti-SMN antibodies (D) or anti-coilin and anti-Flag (for HA-Flag–tagged Sm-D1) antibodies (E). (F) Quantitative results showed the percentage of cells (±SD) in which the indicated proteins colocalized with coilin (n = 3 independent experiments). (G) TOE1 is required for efficient splicing. A splicing reporter was introduced into HeLa cells or WI-38 cells with the indicated treatment. 24 h later, both spliced and unspliced RNAs were amplified from cDNA using the indicated primer sets. (H) The intensity of unspliced and spliced products was quantified by Quantity One software. The ratios of spliced to unspliced RNAs (±SD) were normalized by controls and presented as a bar graph for the indicated groups (n = 3 independent experiments). (I) TOE1 down-regulation suppresses cell growth. Cells were harvested and counted at day 1–5 after siRNA transfection. The cell numbers (±SD) were plotted against the days after siRNA treatment (n = 3 independent experiments). Bars, 10 µm.