Figure S2.
mTORC1 is activated at the cell periphery.(A) HeLa cells grown in full-nutrient medium were FCS starved for 18 h (−FCS) or FCS starved and then recovered in FCS-containing medium for 10 min (10 min FCS). Immunostaining of mTOR and LAMP1 is shown. (B and C) Immunostaining for p-S6 and LAMP1 following starvation and refeeding as in A (B) or in response to amino acid starvation versus amino acid starvation and recovery (C). Arrows indicate mTORC1 activation at the cell periphery. The IntDens of peripheral or intracellular signal of p-S6 were quantified and the colocalization at the cell periphery between p-S6 and LAMP1 was quantified using Manders coefficient. (D) The proportion of cells with peripheral p-S6 staining was quantified in cells starved and recovered in FCS-containing medium (left) or amino acids (right) as indicated. (E) Quantification of the proportion of cells with peripheral LAMP1 in full-nutrient medium that exhibited peripheral p-S6 staining. (F) HeLa cells transfected with FLAG-ARL8B or empty FLAG plasmid were FCS starved for 18 h (−FCS) or FCS starved and then recovered in FCS-containing medium for 10 min (10 min FCS). Immunostaining of p-S6 and LAMP1 (left) or FLAG (right) is shown. The IntDens of peripheral or intracellular signal of p-S6 and the proportion of cells with peripheral p-S6 staining was quantified. (G) Cells were treated as in A, lysed, and subject to immunoblotting for mTORC1 activity (top). For quantification of relative p-S6 levels (bottom), error bars represent SEM; n = 3 independent experiments (for IntDens, n ≥ 10 cells were quantified per experiment). *, P < 0.05; **, P < 0.01; ***, P < 0.001; two-sided Student’s t test. Scale bars, 20 µm (insets, 10 µm). Nuclei were visualized with DAPI.

mTORC1 is activated at the cell periphery. (A) HeLa cells grown in full-nutrient medium were FCS starved for 18 h (−FCS) or FCS starved and then recovered in FCS-containing medium for 10 min (10 min FCS). Immunostaining of mTOR and LAMP1 is shown. (B and C) Immunostaining for p-S6 and LAMP1 following starvation and refeeding as in A (B) or in response to amino acid starvation versus amino acid starvation and recovery (C). Arrows indicate mTORC1 activation at the cell periphery. The IntDens of peripheral or intracellular signal of p-S6 were quantified and the colocalization at the cell periphery between p-S6 and LAMP1 was quantified using Manders coefficient. (D) The proportion of cells with peripheral p-S6 staining was quantified in cells starved and recovered in FCS-containing medium (left) or amino acids (right) as indicated. (E) Quantification of the proportion of cells with peripheral LAMP1 in full-nutrient medium that exhibited peripheral p-S6 staining. (F) HeLa cells transfected with FLAG-ARL8B or empty FLAG plasmid were FCS starved for 18 h (−FCS) or FCS starved and then recovered in FCS-containing medium for 10 min (10 min FCS). Immunostaining of p-S6 and LAMP1 (left) or FLAG (right) is shown. The IntDens of peripheral or intracellular signal of p-S6 and the proportion of cells with peripheral p-S6 staining was quantified. (G) Cells were treated as in A, lysed, and subject to immunoblotting for mTORC1 activity (top). For quantification of relative p-S6 levels (bottom), error bars represent SEM; n = 3 independent experiments (for IntDens, n ≥ 10 cells were quantified per experiment). *, P < 0.05; **, P < 0.01; ***, P < 0.001; two-sided Student’s t test. Scale bars, 20 µm (insets, 10 µm). Nuclei were visualized with DAPI.

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