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. 2017 Aug 7;216(8):2391–2407. doi: 10.1083/jcb.201611108

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© 2017 Prusty et al.

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Figure 1. — SMN knockdown results in mislocalization of SMN complex and UsnRNPs. (A) Lentivirus-mediated knockdown of SMN (shSMN). Western blot analysis of doxycycline-inducible shRNA knockdown of SMN, using tubulin as the normalization control. Quantification of the blot is shown in the bottom panel, with black and gray bars indicating control and SMN knockdown, respectively. (B) FACS analysis of control uninduced cells (top), shSMN at 120 h (middle) and shSMN at 144 h (bottom) after doxycycline induction of shRNA, after staining with annexin V (FL4-A channel) and propidium iodide (FL3-A channel). Left, gating of all cells (brown); right, distribution of necrotic (bottom right quadrant), early apoptotic (top right quadrant), and apoptotic (top left quadrant) cells along with the respective quantification. (C) Western blot using antibodies specific to SMN to validate knockdown in cells used for FACS analysis, with tubulin as loading control. (D) Confocal images of control cells (top) and shSMN cells (bottom) stained using antibodies against SMN, Gemin5, SmD1/B/B'/D3 (Y12 antibody), and coilin as well as DAPI for nuclear staining. White arrowheads in SMN and Gemin5 images indicate predominant staining pattern observed in cytoplasm (shSMN cells) and Cajal bodies (CBs) and cytoplasm (control cells). White arrowheads in Sm images indicate CBs, splicing speckles (control), and speckles (shSMN). White arrowheads in coilin images indicate intact CBs (control) and disintegrated CBs/mislocalized coilin (shSMN).