Figure 3. Protrudin and FYCO1 depletion leads to perinuclear clustering of mTOR-positive lysosomes and decreases mTORC1 activity. (A) RPE-1 cells grown in complete medium were transfected with control RNA or siRNA targeting Protrudin or FYCO1, stained with an antibody to mTOR, and analyzed by high-content wide-field microscopy. (B and C) Automated quantification of images as in A using the Olympus ScanR system, representing the relative ratios of perinuclear/peripheral intensity of mTOR-positive lysosomes. Error bars denote ± SEM from independent experiments (B); n = 3; **, P ≤ 0.01 (one-sample t test); >1,400 cells per condition. (C) siRNA FYCO1 oligo 1 (n = 6), >5,000 cells. siRNA FYCO1 oligo 2 (n = 3), >900 cells. *, P < 0.05; **, P < 0.01 (one-sample t test). (D) Representative immunoblots showing the reduced levels of phosphorylated S6K and 4E-BP1 upon Protrudin depletion in RPE-1 cells grown in complete medium. Graphs represent quantifications from immunoblots. Error bars denote ± SEM from independent experiments. p-S6K (n = 6) or p-4E-BP1 (n = 4). **, P < 0.01; ***, P < 0.001 (one-sample t test). (E) Representative immunoblots showing the reduced levels of phosphorylated S6K and 4E-BP1 upon FYCO1 depletion in RPE-1 cells grown in complete medium. Graphs represent quantifications from immunoblots. Error bars denote ± SEM from independent experiments. p-S6K (n = 4) or p-4E-BP1 (n = 3). **, P < 0.01 (one-sample t test). (F) Representative immunoblots showing the reduced levels of phosphorylated S6K upon Protrudin depletion in HEK293 cells grown in complete medium. Graph represents quantifications from immunoblots. Error bars denote ± SEM from independent experiments (n = 4). *, P < 0.05; **, P < 0.01 (one-sample t test). (G) Representative immunoblots showing the reduced levels of phosphorylated S6K upon FYCO1 depletion in HEK293 cells grown in complete medium. Graph represents quantifications from immunoblots. Error bars denote ± SEM from independent experiments (n = 3). **, P < 0.01 (one-sample t test). (H) Representative immunoblots showing unaffected levels of phosphorylated AKT (mTORC2 site Ser473) upon Protrudin depletion in RPE-1 cells grown in complete medium. Graph represents quantifications from immunoblots. Error bars denote ± SEM from independent experiments (n = 4).
Figure 3.

Protrudin and FYCO1 depletion leads to perinuclear clustering of mTOR-positive lysosomes and decreases mTORC1 activity. (A) RPE-1 cells grown in complete medium were transfected with control RNA or siRNA targeting Protrudin or FYCO1, stained with an antibody to mTOR, and analyzed by high-content wide-field microscopy. (B and C) Automated quantification of images as in A using the Olympus ScanR system, representing the relative ratios of perinuclear/peripheral intensity of mTOR-positive lysosomes. Error bars denote ± SEM from independent experiments (B); n = 3; **, P ≤ 0.01 (one-sample t test); >1,400 cells per condition. (C) siRNA FYCO1 oligo 1 (n = 6), >5,000 cells. siRNA FYCO1 oligo 2 (n = 3), >900 cells. *, P < 0.05; **, P < 0.01 (one-sample t test). (D) Representative immunoblots showing the reduced levels of phosphorylated S6K and 4E-BP1 upon Protrudin depletion in RPE-1 cells grown in complete medium. Graphs represent quantifications from immunoblots. Error bars denote ± SEM from independent experiments. p-S6K (n = 6) or p-4E-BP1 (n = 4). **, P < 0.01; ***, P < 0.001 (one-sample t test). (E) Representative immunoblots showing the reduced levels of phosphorylated S6K and 4E-BP1 upon FYCO1 depletion in RPE-1 cells grown in complete medium. Graphs represent quantifications from immunoblots. Error bars denote ± SEM from independent experiments. p-S6K (n = 4) or p-4E-BP1 (n = 3). **, P < 0.01 (one-sample t test). (F) Representative immunoblots showing the reduced levels of phosphorylated S6K upon Protrudin depletion in HEK293 cells grown in complete medium. Graph represents quantifications from immunoblots. Error bars denote ± SEM from independent experiments (n = 4). *, P < 0.05; **, P < 0.01 (one-sample t test). (G) Representative immunoblots showing the reduced levels of phosphorylated S6K upon FYCO1 depletion in HEK293 cells grown in complete medium. Graph represents quantifications from immunoblots. Error bars denote ± SEM from independent experiments (n = 3). **, P < 0.01 (one-sample t test). (H) Representative immunoblots showing unaffected levels of phosphorylated AKT (mTORC2 site Ser473) upon Protrudin depletion in RPE-1 cells grown in complete medium. Graph represents quantifications from immunoblots. Error bars denote ± SEM from independent experiments (n = 4).

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