Figure S5.
Disruption of FAs or impairment of peripheral lysosomal distribution inhibits mTORC1 activation by nutrients.(A and B) HeLa cells transfected with scrambled (Scr) or TLN1/2 siRNAs grown in full-nutrient medium were amino acid and FCS starved for 1 h (−aa −FCS) and then recovered in amino acid–containing medium (10 min aa) or amino acid– and FCS-containing medium (10 min aa +FCS). Cells were analyzed by immunostaining for LAMP1 and p-S6 (A) or immunoblotting to detect mTORC1 activity (B). (C) HeLa cells subjected to amino acid and FCS starvation for 1 h were scraped (suspension) or left adherent and then refed as described in A and subjected to immunoblot analysis to detect mTORC1 activity. (D and E) HeLa cells were FCS starved for 18 h (−FCS); treated with DMSO (control), ROCKi, or integrin antagonist (cilengitide) for 1 h in −FCS medium; starved of amino acids (−aa −FCS) for 1 h in the presence of inhibitors; and then recovered in full-nutrient medium for 10 min. Cells were subjected to immunoblot analysis to detect mTORC1 activity (D). The IntDens of peripheral paxillin staining was quantified (E). (F) Scrambled (Scr) or ARL8B siRNA–transfected HeLa cells were FCS starved for 18 h (−FCS) and then recovered in FCS-containing medium for 10 min (10 min FCS). Immunostaining of p-S6 and LAMP1 is shown. (G) HeLa cells expressing FLAG-RPTOR and FLAG-RPTORFAT grown in full-nutrient media were lysed, immunoprecipitated with FLAG antibody, and immunoblotted for FLAG and mTOR (left) and FLAG and paxillin (right). (H) Fluorescence intensity line profile plots corresponding to lines exemplified by arrows in Fig. 6 A are shown. (I) Scrambled or ARL8B siRNA–transfected HeLa cells were FCS starved for 18 h (−FCS) and then recovered in FCS-containing medium for 10 min (10 min FCS). Immunostaining for paxillin (left) and fluorescence intensity line profile plots corresponding to lines exemplified by arrows (right) are shown. (J) Scrambled or ARL8B siRNA–transfected HeLa cells expressing FLAG-RPTOR or FLAG-RPTORFAT grown in full-nutrient medium were FCS starved for 18 h (−FCS) or FCS starved and then recovered in FCS-containing medium for 10 or 60 min (10, 60 min FCS), lysed, and subjected to immunoblot analysis using antibodies as shown. Error bars represent SEM; n = 3 independent experiments. For J, n = 2 independent experiments and error bars represent SD. *, P < 0.05; **, P < 0.01; two-sided Student’s t test (performed between groups). Scale bars, 20 µm (insets, 10 µm). Nuclei were visualized with DAPI.

Disruption of FAs or impairment of peripheral lysosomal distribution inhibits mTORC1 activation by nutrients. (A and B) HeLa cells transfected with scrambled (Scr) or TLN1/2 siRNAs grown in full-nutrient medium were amino acid and FCS starved for 1 h (−aa −FCS) and then recovered in amino acid–containing medium (10 min aa) or amino acid– and FCS-containing medium (10 min aa +FCS). Cells were analyzed by immunostaining for LAMP1 and p-S6 (A) or immunoblotting to detect mTORC1 activity (B). (C) HeLa cells subjected to amino acid and FCS starvation for 1 h were scraped (suspension) or left adherent and then refed as described in A and subjected to immunoblot analysis to detect mTORC1 activity. (D and E) HeLa cells were FCS starved for 18 h (−FCS); treated with DMSO (control), ROCKi, or integrin antagonist (cilengitide) for 1 h in −FCS medium; starved of amino acids (−aa −FCS) for 1 h in the presence of inhibitors; and then recovered in full-nutrient medium for 10 min. Cells were subjected to immunoblot analysis to detect mTORC1 activity (D). The IntDens of peripheral paxillin staining was quantified (E). (F) Scrambled (Scr) or ARL8B siRNA–transfected HeLa cells were FCS starved for 18 h (−FCS) and then recovered in FCS-containing medium for 10 min (10 min FCS). Immunostaining of p-S6 and LAMP1 is shown. (G) HeLa cells expressing FLAG-RPTOR and FLAG-RPTORFAT grown in full-nutrient media were lysed, immunoprecipitated with FLAG antibody, and immunoblotted for FLAG and mTOR (left) and FLAG and paxillin (right). (H) Fluorescence intensity line profile plots corresponding to lines exemplified by arrows in Fig. 6 A are shown. (I) Scrambled or ARL8B siRNA–transfected HeLa cells were FCS starved for 18 h (−FCS) and then recovered in FCS-containing medium for 10 min (10 min FCS). Immunostaining for paxillin (left) and fluorescence intensity line profile plots corresponding to lines exemplified by arrows (right) are shown. (J) Scrambled or ARL8B siRNA–transfected HeLa cells expressing FLAG-RPTOR or FLAG-RPTORFAT grown in full-nutrient medium were FCS starved for 18 h (−FCS) or FCS starved and then recovered in FCS-containing medium for 10 or 60 min (10, 60 min FCS), lysed, and subjected to immunoblot analysis using antibodies as shown. Error bars represent SEM; n = 3 independent experiments. For J, n = 2 independent experiments and error bars represent SD. *, P < 0.05; **, P < 0.01; two-sided Student’s t test (performed between groups). Scale bars, 20 µm (insets, 10 µm). Nuclei were visualized with DAPI.

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