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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 2. SMN depletion reduces autophagic flux.

Figure 2

NSC-34 #4-#56 cells were treated with doxycycline for 48 hrs to induce SMN knockdown, and then transfected with mCherry-eGFP-LC3. Control and SMN-depleted cultures were treated with rapamycin to induce autophagy and live-cell confocal micrographs were captured to visualize total autophagosomes (yellow) compared to autophagosomes that had successfully fused with the lysosome (red). A) SMN knockdown cells display significantly increased number of yellow puncta (merged image) compared to controls (p=0.027 by Student’s t-test), indicating a reduced rate of autophagic flux (quantified in B). C) Western blot analysis demonstrates increased p62 and LC3-II, which is restored to normal by infection with lentivirus expressing FLAG-hSMN. D) Quantitative analysis of repeated Western blots shows a statistically significant increase in p62 and LC3-II protein levels by Student’s t-test following SMN knockdown. E) Staining for endogenous p62 in SMN-depleted cultures shows an increase in the number of cells with bright p62 puncta (quantified in F, p<0.05 by Student’s t-test).