Figure 6

EB-1/AIDA-1 siRNA disrupts Cajal body structure and causes cell death. (A) Endogenous EB-1/AIDA-1 protein levels are reduced upon treatment with EB-1/AIDA-1 siRNA. HeLa cells were treated with EB-1/AIDA-1 siRNA (100 nM) for 72 hours, followed by nuclear extraction and Western blotting using anti-AIDA-1 antibodies. The film with the 47 kDa. protein was exposed for a shorter period of time. The blot was reprobed with anti-SMN to verify that equivalent levels of extract were loaded on the gel. (B) Coilin and snRNP localization is disrupted upon treatment with actinomycin D. Panel A shows the normal localization of coilin in CBs (arrows), along with the DAPI stain of the same cell. Panel B shows the normal localization of snRNPs to CBs (arrows) and speckles (arrowheads). Panels C and D are representative images of cells treated with actinomycin D. Note that coilin is relocalized to the periphery of the nucleolus (panel C, arrows) and speckles are enlarged (panel D, arrowheads) in the presence of actinomycin D. (C) HeLa cells exposed to EB-1/AIDA-1 siRNA (100 nM for 72 hours) were subjected to immunofluorescence analysis with antibodies to coilin (red) and snRNPs (Y12) (green). The overlay is shown in the far right panel. Arrows mark altered Cajal body structure. The bottom panels show a closer view of another cell with disrupted CB organization and co-localization of coilin and snRNPs.