FIVeGFP.shRNA-mediated silencing of torsinA(ΔE) prevents formation of spheroid bodies. A , Western blot analysis of PC6-3 torsinA(ΔE)-expressing cell lines, showing potent suppression of torsinA(ΔE) expression by FIVeGFP.shTAcom and shTAmut5 but not a FIVeGFP control. The formation of a higher molecular weight, torsinA-immunoreactive band of ∼85 kDa (arrow), was also suppressed. GFP, FIVeGFP; com, FIVeGFP.shTAcom; mut5, FIVeGFP.shTAmut5. B , Quantification of results from three experiments as in A . C , Immunofluorescence image, with GFP as a reporter for transduced cells, showing prevention of spheroid body formation by FIVeGFP.shTAmut5 but not by FIVeGFP. Blue, DAPI nuclear staining. D , Quantification of the presence of spheroid bodies in three independent experiments in nontransduced control cells or cells transduced by different viral vectors. Error bars indicate SE. DOX, Doxycycline; com, U6shTAcom; mut5, U6shTAmut5; tub, α-tubulin; WB, Western blot; torA, torsinA.
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