Figure 6. HeLa cells have constitutive lysosomal TSC2 localization even under basal culture conditions.

(a,b) In HeLa cells, TSC2 is concentrated on lysosomes in basal, serum-containing conditions and only relocalizes to the cytoplasm upon hyperstimulation with insulin (1 μM). (c,d) In HeLa cells, TSC2 constitutively accumulates on lysosomes in medium containing serum and amino acids. Since amino-acid levels present in DMEM are not sufficient to dislodge TSC2 from lysosomes in HeLa cells, TSC2 is lysosomally accumulated both in basal culture conditions (top row, +aa), and when amino acids are first removed and then restored to the starting concentrations (bottom row). (e,f) The re-addition (re-add.) of both amino acids and high insulin are required to blunt TSC2 lysosomal localization in doubly starved HeLa cells. HeLa cells were starved for both FBS (16 h) and amino acids (1 h), and then either insulin (1 μM, 15 min), +aa media (30 min) or both were added back to the cells. Note the presence of lysosomal TSC2 even upon high insulin re-stimulation (−FBS −aa→+Ins), which is only blunted when amino acids are also added back, indicating that both insulin signalling and amino acids are required for cytoplasmic localization of TSC2. For a,c and e TSC2 localization was assayed in HeLa cells as in Fig. 1, and the degree of co-localization between TSC2 and LAMP2 (automatically thresholded MCC) is shown in b,d and f as mean±s.e.m. ***P<0.001, **P<0.01 for comparison to control (Ctrl) or as indicated, using one-way ANOVA. For all panels, images representative of at least three independent biological replicates are shown. See also Supplementary Figs 11–16.