This was accomplished by employing a siRNA that targets the 39 untranslated region of the BIBW2992 cost endogenous coilin message. Since the 39 UTR was deleted in the constructs used to generate the stable cell lines, treatment of cells with this siRNA reduces endogenous coilin but does not affect the expression of the various GFP-coilin proteins . It should be noted, however, that reduction of endogenous coilin and subsequent induction of the respective GFP-coilin protein results in approximately equal amounts of endogenous coilin with the GFP-coilin protein. For example, since the level of GFP-coilin WT upon induction is lower than that of endogenous coilin, knockdown of endogenous coilin results in roughly equal amounts of GFP-coilin WT to endogenous coilin . Consequently, any observed changes in CB number and proliferation are indicative of relatively small changes in the ratio of endogenous coilin to the various GFP-coilin proteins, which makes this system more physiologically relevant than that found when conducting transient transfections. Immunofluorescence analysis was conducted on each cell line first treated with control or coilin siRNAs and then exposed to doxycycline to induce the expression of the various GFP-tagged coilin proteins. Amongst the different cell lines, only WT is capable of forming robust CBs that contain SMN in most cells upon endogenous coilin knockdown . The coilin phosphomutants, in 391210-10-9 contrast, all had reduced CB formation potential in cells treated with coilin-siRNA compared to controlsiRNA. Notably, most of the ON expressing cells in the coilin knockdown background were nucleoplasmic and lacked CBs, but contained SMN foci . Cell lines that contain numerous CBs upon exposure to control siRNA, such as S271/272D and OFF, showed a significant decrease in CB number after endogenous coilin knockdown . Additionally, the frequency of coilin foci lacking SMN and Gems were increased in the S271/272D cell line upon coilin knockdown . T122E expression in cells treated with coilin siRNA generated some CBs , but also resulted in coilin foci that did not efficiently recruit SMN, which accumulated in Gems . More drastic changes were observed for S489D upon coilin siRNA treatment . In control treated cells, most cells have accumulations of this protein in CBs and nucleoli , identical to that observed without siRNA treatment after induction . However, in coilin siRNA treated cells, the nucleolar accumulation of S489D is less discrete and the foci formed by GFP-coilin do not accumulate SMN .