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. Author manuscript; available in PMC: 2015 Jul 1.
Published in final edited form as: Mol Cell Neurosci. 2014 Jun 28;0:133–140. doi: 10.1016/j.mcn.2014.06.006

Figure 1. SMN knockdown in NSC-34 cells induces autophagy.

Figure 1

Clonal NSC-34 cells expressing a doxycycline-inducible shRNA against murine SMN (clone #4-56) were treated with doxycycline (2ug/ml) for 48 hours to deplete SMN levels to 47.4 +/− 10.9% relative to controls (inset in A shows Western blot of SMN levels following 48hr knockdown) and then transfected with GFP-LC3 to visualize autophagosomes. A) Control cultures show mostly diffuse LC3-GFP fluorescence with very few autophagic puncta per cell compared with Dox-treated cultures, which accumulate numerous GFP-positive autophagic puncta. B) Quantification of the number of LC3-GFP puncta per cell shows a that SMN-depleted cells have a statistically significant increase in puncta by Student’s t-test compared to control cultures (p<0.05). No increase in autophagic puncta was observed when cells from clone #4, which express only the rTta plasmid, were treated with doxycycline. C) Western blotting for LC3 shows that following SMN knockdown, the ration of LC3-II/LC3-I increases. Removal of doxycycline for 48 hrs allows SMN levels and LC3 ratios to return to normal. D) Quantitative analysis of repeated Western blots shows a significant increase in LC3-II protein following SMN depletion (p<0.05 by Student’s t-test).