1. Fig. 5 C9orf72 knockdown disrupts autophagy induction.
C9orf72 knockdown disrupts autophagy induction. (A) Western blot analysis of LC3 expression. MEF cells infected with lentiviruses expressing control or C9orf72 shRNA were treated with DMSO or rapamycin (0.1 μM) for 3.5 hours before protein lysate collection. β-Actin serves as the loading control. (B) Quantification of LC3-II/actin ratio. Error bars represent SEM of three measurements from three independent experiments; *P < 0.05 (Student’s t test). n.s. represents no significant difference in LC3-II expression between DMSO and rapamycin treatment in C9orf72 knockdown MEFs. Two-way ANOVA detects a significant decrease in the magnitude of LC3-II up-regulation after rapamycin treatment in the C9orf72 knockdown MEFs compared to that in wild-type MEFs (P < 0.05). (C) Confocal imaging of MEFs stained with antibodies against LC3 (green). MEF cells were infected with lentiviruses expressing control or C9orf72 shRNA followed by starvation. Hoechst stains the nuclei (blue). Scale bars, 10 μm. (D) Quantification of LC3-positive puncta per cell in (C). Error bars represent SEM of three independent experiments; ~100 cells were randomly selected for each experiment. *P < 0.05 (Student’s t test). (E) Western blot analysis of LC3 expression. Wild-type or Smcr8 mutant MEF cells were infected with lentiviruses expressing control or C9orf72 shRNA as indicated followed by DMSO or rapamycin (0.1 μM) treatment for 3.5 hours before protein lysate collection. β-Actin serves as the loading control. (F) Quantification of LC3-II/actin ratio. Error bars represent SEM of three measurements from three independent experiments; *P < 0.05 (Student’s t test). n.s. represents no significant difference in LC3-II expression between DMSO and rapamycin treatment. Two-way ANOVA detects a significant decrease in the magnitude of LC3-II up-regulation after rapamycin treatment in the C9orf72 knockdown, Smcr8 mutant, and C9orf72/Smcr8 double depletion MEFs compared that in to wild-type MEFs (P < 0.05).
Mei Yang et al. Sci Adv 2016;2:e
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