Protrudin is required for amino acid–dependent lysosome redispersion after starvation. (A) RPE-1 cells were grown in complete medium, starved with EBSS for 4 h, and stimulated or not with 2× amino acids (AA) in DMEM with or without FCS for 2 h, stained with an antibody to LAMP1, and imaged by wide-field microscopy. (B) RPE-1 cells grown in complete medium were transfected with control RNA or siRNA targeting Protrudin, starved with EBSS for 4 h, and stimulated or not with 1× amino acids in DMEM for 1 h, stained with an antibody to LAMP1, and imaged by high-content wide-field microscopy. (C) Automated quantification of images as in B using the Olympus ScanR system, representing the relative ratios of peripheral/perinuclear intensity of LAMP1-positive lysosomes. Error bars denote ± SEM from independent experiments (n = 3). *, P < 0.05; **, P < 0.01. Treatments versus fed control set to 1 (one-sample t test), samples with equal variance (unpaired t test). Greater than 1,900 cells were analyzed per condition.