Fig. 5: Pharmacological inhibition of mTORC1 activity using rapamycin enhances lysosomal localization and function.

a Immunoblot analysis of lysates from COS-7 cells that were transfected with siCtrl/siUBAP1 for 48 h, and incubated with/without 500 nM Rapamycin (Rap) or 100 nM BafA1 for 5 h, and probed with indicated antibodies. Quantification of relative expression, normalized to GAPDH. All groups n = 4 independent samples. b Left: COS-7 cells were transfected with siCtrl/siUBAP1 and LAMP1-GFP for 48 hours and incubated with/without 500 nM Rapamycin (Rap) for 24 h, and stained with antibody against mTOR. Right: Colocalization analysis of mTOR with LAMP1 in COS-7 cells. Mock: siCtrl n = 17, siRNA n = 19; Rap: siCtrl n = 20, siRNA n = 20 cells. c COS-7 cells were transfected with siCtrl/siUBAP1 and LAMP1-RFP for 48 h and incubated with/without 500 nM Rapamycin (Rap) for 24 h, staining with antibody against p62. Quantification of the ratio of lysosomes positive for both p62 and LAMP1. Mock: siCtrl n = 12, siRNA n = 12; Rap: siCtrl n = 12, siRNA n = 12 cells. d Left: COS-7 cells were transfected with siCtrl/siUBAP1 and LAMP1-mYH for 48 h and incubated with/without 500 nM Rapamycin (Rap) for 5 h. Right: Quantification of the inner diameter of lysosomes. Mock: siCtrl n = 218, siRNA n = 239; Rap: siCtrl n = 592, siRNA n = 550 lysosomes. All scale bars: 10 μm. All data represent the mean ± SEM; Student’s t test (a), Two-way ANOVA followed by Sidak’s multiple comparisons test (b, c), Kruskal–Wallis test followed by Dunn’s multiple comparisons test (d); ns p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ^#p < 0.0001. Exact p-values and test statistics are provided as a Source Data file.