Ragulator regulates lysosome positioning independently of mTORC1. (A) HeLa cells stably transfected with nontargeting (control), LAMTOR1, or RAPTOR shRNAs and cultured in regular medium were analyzed by SDS-PAGE and immunoblotting for phosphorylation (p) of the mTORC1 substrate S6K and levels of LAMTOR1, LAMTOR2, and RAPTOR. The positions of molecular mass markers (in kilodaltons) are indicated on the left. (B) mTOR localization and lysosome distribution in the cells in A was visualized by immunostaining with antibodies to mTOR and LAMP1. Bar, 10 µm. (C) Lysosome distribution in the cells in B (15 cells from three independent experiments for each cell line) was quantified by measuring lysosome-to-MTOC distance using Imaris software and shown as box-and-whisker plots. The ends of whiskers represent the minima and maxima of the data. ****, P < 0.0001; n.s., not significant (ANOVA). (D) HeLa cells were treated with the mTOR inhibitors PP242 (200 nM), rapamycin (2 µM), KU-0063794 (1 µM), or Torin1 (200 nM) or with DMSO (control), for different times at 37°C, and phosphorylation of the indicated mTORC1 substrates was analyzed by SDS-PAGE and immunoblotting. The positions of molecular mass markers (in kilodaltons) are indicated on the left. (E) HeLa cells were treated with mTOR inhibitors for 2 h at 37°C as in D and immunostained for LAMP1. Bar, 10 µm. (F) Quantification of lysosome distribution from 20 cells in three independent experiments such as that in E using Imaris. Data are shown as box-and-whisker plots with minimum and maximum range. n.s., not significant (ANOVA).