Fig 1: Generation of Ragulator-deficient clone with total mTORC1 activity.A, schematic representation of the findings of this study, depicting the phosphorylation of the TFEB substrate by mTORC1 bound to the Ragulator-Rag scaffold. B, the results of Western blotting studies on ΔRagulator and parental RAW264.7 cells. Lamtor1, Lamtor2, Lamtor3, Lamtor4, and Lamtor5 are components of the Ragulator complex; β-actin served as the internal control; mTOR and Raptor are components of mTORC1; RagC is necessary for the docking of TFEB to Ragulator. C, representative result of Western blotting studies for determining the activity of mTORC1. The control RAW264.7 cells and ΔRagulator cells were cultured in nutrient-sufficient and serum-supplemented DMEM. The phosphorylation of S6K in these cells was abolished following treatment with the mTOR kinase inhibitor, Torin1, at the indicated concentrations for 1 h. D, the densities of the p-S6K and S6K bands in panel C were quantified. The ratio of densities of the p-S6K band to S6K band is shown. Four independent experiments were performed. Bars indicate the median. Statistical significance was calculated using Mann–Whitney U test. E, the results of Upstream Regulator Analysis, as determined using the web-based software, Ingenuity Pathway Analysis. The pathways with z scores less than −2 are significantly inactivated in the ΔRagulator cells, compared to the RAW264.7 cells. The z score for the TFEB pathway was >2, which indicates that TFEB is significantly activated in the ΔRagulator cells than in the control RAW264.7 cells. F, the sizes of the ΔRagulator cells were larger than those of the parental RAW264.7 cells. The cell sizes of 1 × 104 cells were estimated using flow cytometry. Statistical significance was calculated using Welch’s t test. G, Western blotting for determining the phosphorylation of TSC2. Control RAW264.7 cells and ΔRagulator cells were cultured in nutrient-sufficient and serum-supplemented DMEM. Three biological triplicates were examined. H, the densities of the p-TSC2 and the TSC2 bands in panel G were quantified. The ratio of densities of the p-TSC2 band to TSC2 band is shown. Statistical significance was calculated using the Mann–Whitney U test. mTORC1, mammalian target of rapamycin complex 1; TFEB transcription factor EB.