Journal: Cell Communication and Signaling : CCS
Article Title: Peripheral lysosome levels dictate mTORC1 inactivation even when catabolically impaired
doi: 10.1186/s12964-026-02659-9
Figure Lengend Snippet: Rap1 activity is required for peripheral lysosome relocation and sustained mTORC1 suppression during prolonged leucine starvation. A–D Effects of Rap1 depletion on lysosome organization under amino acid-fed and prolonged leucine deprivation. U2OS ( A , B ) or HEK293A ( C , D ) cells were transfected with control or Rap1A + B siRNA and cultured in amino acid-fed ( A , B ) or leucine-free ( C , D ) medium . Representative z-stack confocal maximum-intensity projections ( A ) and fluorescence images ( C ) are shown, with quantifications of lysosome peripheral distribution in ( B , D ). E, F Immunoblot analysis of mTORC1-associated signaling in Rap1-depleted HEK293A cells starved for the indicated time points. G Representative single confocal z-slices of mTOR and LAMP2-marked lysosomes in HEK293A cells transfected with Rap1A + B siRNA under amino acid-fed or leucine-starved conditions, with quantifications of mTOR/LAMP2 colocalization in ( H ). I Immunoblot analysis of mTORC1 signaling in HEK293A cells expressing EGFP-Rab7 WT or EGFP-Rab7 Q67L . All experiments were repeated at least three times. Scale bars: ( A , C , G ) = 10 μm. dFBS was included in all leucine starvation experiments. In ( B , D ), "n" denotes the number of individual cells analyzed across 3 independent experiments, data are presented as mean values ± s.d.; with statistical analysis performed using one-way ANOVA with Tukey’s post hoc test. Immunoblot quantifications across at least three individual experiments are presented as mean ± SEM and were statistically analyzed using two-tailed unpaired Student’s t-test ( n.s., not significant; p ≥ 0.05). In ( B ), outliers were detected and removed using the 1.5× Interquartile Range (IQR) method before statistical testing
Article Snippet: For multiplex fluorescence blotting, membranes were incubated in IRDye 680RD or 800CW (LI-COR) and imaged with an Odyssey Fc Imager (LI-COR).
Techniques: Activity Assay, Transfection, Control, Cell Culture, Fluorescence, Western Blot, Expressing, Two Tailed Test
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