EGF regulates LAMTOR–BORC interaction. (A) The interaction between BORC and LAMTOR complexes is decreased upon EGF stimulation. Lyspersin KO cells stably expressing HS-lyspersin were starved for FBS and stimulated with 100 ng/ml EGF for 1 h. Obtained lysates were Strep-purified and analyzed by immunoblotting. EGFR degradation was used as control for the treatment. (B) Quantification of the results of three biologically independent experiments. Mean ± SD. ***, P ≤ 0.001; P = 0.000352. (C) Confirmation that the interaction between BORC and LAMTOR complexes is decreased upon EGF stimulation with HEK293 Flp-In T-REx cells inducibly expressing diaskedin-SH starved and stimulated as in A. Immunoblotting analysis of the Strep-purified complexes. EGFR degradation was used as control for the treatment. (D) Quantification of the results of three biologically independent experiments. Mean ± SD. ***, P ≤ 0.001; P = 0.000938. (E) Arbitrary cutoff for peripheral late endosomes based on LAMP1-positive vesicles distribution in starved WT HeLa cells. Epifluorescence images were analyzed using ImageJ software with our macro RadialIntensityProfile (Materials and methods). Depicted is the signal intensity of LAMP1 found at the indicated distance from the center of nucleus, perinuclear lysosomes ≤16 µm, peripheral lysosomes ≥16 µm. Number of cells per genotype, n ≥ 50 cells. (F) Frequency of peripheral lysosomes increases upon EGF stimulation. WT HeLa cells were starved for FBS overnight and stimulated with EGF for the indicated time points and subsequently treated as in E. Depicted is the signal intensity of LAMP1 found at ≥16 µm from the center of the nucleus. Mean ± SD. Not significant (n.s.), P > 0.05; ****, P ≤ 0.0001 (0 h/1 h P = 0.630, 0 h/4 h P = 1.1 × 10^-8, 0 h/8 h P = 3.2 × 10^-7). Number of cells per genotype, n ≥ 50 cells.