Fig 6. C9orf72 regulates TFEB translocation from the nucleus to the cytoplasm upon amino acid stimulation.

(A) Representative immunostaining images of HeLa cells stably expressing TFEB-GFP, with C9orf72 or control shRNA knockdown, after amino acid stimulation for 10 min following a period of 50 min of amino acid starvation. Immunostaining with anti-GFP and an Alexa488-conjugated secondary antibody was performed together with DAPI staining. Scale bar: 20 μm. (B) Quantification of TFEB-GFP subcellular localization in (a). The numbers of cells carrying nuclear or cytoplasmic TFEB-GFP were quantified. [*p<0.05, n = 19 for control shRNA-expressing cells and n = 11 for C9orf72-specific shRNA-expressing cells]. Distribution of data points is presented with mean ± SD. (C) Decreased phosphorylated TFEB level was rescued by expression of an active forms of Rag GTPases when the starved cells were stimulated with amino acids. Total TFEB was immunoprecipitated and phosphorylated TFEB was detected by western blotting in the precipitates.