Figure 2.

Nuclear translocation of TFEB occurs in Ragulator-deficient cells, regardless of total mTORC1 activity.A, representative result of Western blotting for determining the activity of mTORC1 and subcellular distribution of TFEB. The nuclear translocation of TFEB increased in the ΔRagulator and RAW264.7 cells after 1 h of amino acid starvation and treatment with 250 nM of Torin1. The internal controls for the cytosolic and nuclear fractions were β-actin and Lamin B, respectively. The quantities of S6K, phospho-S6K, and Lamtor1 were determined from the cytosolic fraction, which contained the lysosomes. B, the density of the TFEB band in panel A was quantified. The ratio of nuclear to cytosolic TFEB is shown. Four independent experiments were performed. Bars indicate the median. Statistical significance was calculated using the Mann-Whitney U test. C, gene expression microarray revealed that expression levels of RagC and RagD genes in ΔRagulator cells were higher than those in control RAW264.7 cells. D, the intensity of SSC for the ΔRagulator and RAW264.7 cells were obtained by flow cytometric analysis of 1 × 10⁴ cells. A high intensity of SSC is indicative of increased intracellular complexity, such as an increase in the numbers of organelles. Statistical significance was calculated using Welch’s t test. E, the images obtained by transmission electron microscopy of ΔRagulator cells and parental RAW264.7 cells are depicted. The cells were cultured in nutrient-sufficient and serum-supplemented DMEM for 1 day, and then fixed immediately. The representative images of the cells obtained by transmission electron microscopy are provided. The yellow arrows indicate the lysosomes. The scale bar represents 2 μm. F, the number of lysosomes and mitochondria in the cells were quantified from the images obtained by electron microscopy. Each of the dots represents a single cell. Statistical significance was calculated using the Mann–Whitney U test. G, immunoprecipitation using the anti-FLAG antibody. A RAW264.7 clone that stably expressed the Lamtor1-3×FLAG protein was used for the immunoprecipitation studies, along with the parental RAW264.7 cells. The high-molecular-weight Lamtor1 protein in the input sample was Lamtor1-3×FLAG, while the low-molecular-weight band represents the endogenous Lamtor1. The results proved the occurrence of a mechanistic association between TFEB and Lamtor1. DMEM, Dulbecco’s modified Eagle’s medium; mTORC1, mammalian target of rapamycin complex 1; TFEB transcription factor EB.