Loss of C9orf72 Enhances Autophagic Activity via Deregulated mTOR and TFEB Signaling
Fig 7
SMCR8 regulates mTOR signaling and autophagy.
A) Immunoblot analysis of mTOR activity after starvation and amino acid stimulation. HAP1 control and SMCR8 KO cells were starved for 50 minutes and supplemented with amino acids for 10–20 min before lysate collection. The mTOR activity was assessed via immunoblotting for the phosphorylation of its downstream substrate S6K1. B) Quantification of p-SK61 (T389) levels after starvation and amino acid stimulation in control and SMCR8 knockout cells from three independent experiments. Knockout of SMCR8 significantly decreased p-S6K1 levels compared with wild-type cells (n = 3, *p<0.05, **p<0.005). C) Immunoblot analysis of LC3 levels after shRNA-mediated knockdown of SMCR8 in HEK293T cells. HEK293T cells were transfected with SMCR8 or control shRNA and lysates collected 72 hours after transfection. The indicated proteins were detected by immunoblotting of the lysates using an antibody against LC3. D) Quantification of the LC3II to LC3I ratio obtained after shRNA-mediated knockdown of SMCR8 in HEK293T cells from three independent experiments. Knockdown of SMCR8 significantly decreased the ratio of LC3II to LC3I compared with control cells (n = 3, *p<0.05). E) Immunoblot analysis of LC3 levels before and after autophagy induction with nutrient starvation. HEK293T cells were transfected with SMCR8 shRNA or scrambled shRNA control. Approximately 72 hours after transfection, cells were treated with starvation medium (EBSS) with and without Bafilomycin for 2 hours and the resulting lysates were analyzed via immunoblotting for LC3. Student’s t test is used and data is presented as mean ± SEM.