Fig. 1: mTORC1 inhibition leads to SCYL1 redistribution to peripheral cytoplasmic locations.

a SCYL1 localizes to cell periphery upon mTORC1 inhibition. Endogenous SCYL1 puncta localize further away from nuclei upon rapamycin treatment. Representative fluorescence microscopy images from MCF-7, HeLa and A549 cells in untreated (DMEM) and Rapamycin-treated (Rapa, 3 h, 100 nM) conditions. One representative image out of n > 3 biological replicates is shown. Scale bar = 10 µm. See also Supplementary Fig. 1. b Quantification of SCYL1 puncta distribution shown in a. Distances of individual SCYL1 puncta from the closest nuclear envelope were averaged per image and their relative distribution is shown. An unpaired two-tailed Student’s t-test was used to compare DMEM and Rapa-treated cells: ****p ≤ 0.0001 (MCF-7: p = 1.25e-19; HeLa: p = 2.87e-68; A549: p = 0.84). n = 3 biological replicates, with a minimum of 10 cells measured per replicate. Error bars, SEM; ns: not significant. c The COPI-coat complex redistributes with SCYL1 to cytoplasmic locations in mTORC1-inhibited conditions. Fluorescence micrograph from WT MCF-7 cells untreated (DMEM) or treated for 3 h with 100 nM Rapa. Yellow lines: line drawings used to generate plot profiles in d. Representative pictures of n = 3 biological replicates are shown. Scale bar = 10 µm. d Plot profiles of c. e SCYL1 distance to Golgi increases upon Rapamycin treatment. SCYL1 puncta distance to GM130-positive Golgi edge as in c was measured and compared between DMEM and 3 h Rapamycin-treated conditions; unpaired two-tailed Student’s t-test, ****: p = 3.91E-181. n = 8 biological replicates for DMEM and n = 7 for Rapa (SCYL1 foci from >10 cells measured per replicate). Error bars: SEM. f Co-localization quantification of c. An unpaired two-tailed Student’s t-test was used to compare co-localization changes (SCYL1/GM130: p = 0.14 (ns); SCYL1/COPA: p = 0.0038 (**)). n = 4 biological replicates. Error bars: SEM. g Endosomal SCYL1 increases upon mTORC1 inhibition. Cellular fractionation of MCF-7 cells untreated (DMEM) or treated for 3 h with 100 nM Rapa or starved in HBSS (Starv). ConA was added in parallel to all conditions to block lysosomal degradation. Proteins within each fraction were extracted and quantified, and equal amounts were loaded per well. WCL: Whole-cell lysates; 17k: 17,000 x g fractionation pellet enriched in endosomal membranes. One representative experiment out of n = 3 biological replicates is shown. h Quantification of g. n = 3. p-values: unpaired two-tailed Student’s t-test value of the indicated data point, compared with the reference–value “DMEM-conA”. WCL SCYL1 protein levels were normalized to ß-Actin/ACTB levels, whereas 17 K SCYL1 protein levels were normalized to EEA1 levels. p-values: SCYL1/WCL, DMEM + conA p = 0.044 (*); Rapa + conA p = 0.011 (*). SCYL1/17 K: DMEM + conA: p = 0.0073 (**); Rapa – conA: p = 0.045 (*); Rapa + conA: p = 0.00044 (***); Starv – conA: p = 0.0021 (**); Starv + conA: p = 0.0058 (**). RAB7/17 K: Rapa + conA: p = 0.012 (*)). Error bars: SEM. i SCYL1 co-localization with early and late endosome markers RAB5 and RAB7 increases upon mTORC1 inhibition. Fluorescence micrograph from untreated WT MCF-7 cells (DMEM) or treated for 3 h with 100 nM Rapamycin (“Rapa”). Representative pictures of n = 3 replicates are shown. Scale bar = 10 µm. j Co-localization quantification of i. n = 2 biological replicates for SCYL1/RAB5 and SCYL1/LAMP2 measurements, and n = 3 for SCYL1/RAB7 measurements. An unpaired two-tailed Student’s t-test was used to compare SCYL1/RAB7 co-localization changes between DMEM and Rapa-treated conditions (p = 0.025 (*)). Ten cells were measured per slide, and three slides averaged per biological replicate. Error bars: SEM. All source data are provided as Source Data File.