Figure 7. Lysosomal perturbations cause GLUT1 mis-sorting.

(A, B) High content microscopy (HCM) imaging and quantification of LC3 response (puncta/cell of endogenous LC3 immunofluorescent profiles) in HeLa^WT, and HeLa^VPS35-KO cells in response to lysosomal damage by LLOMe (1 mM, 30 and 60 min). Scale bar, 20 μm. Data, means ± SE (n = 5), one-way ANOVA with Tukey’s multiple comparisons. (C–E) HCM quantification of GLUT1 response (puncta/cell of endogenous GLUT1) (D) and its localization to endolysosomal compartments (% of LAMP1 profiles positive for GLUT1 immunostaining) (E) in Huh7^WT treated with or without Monensin (100 µM), LLOMe (100 µM), and Bafilomycin A1 (100 nM) for 45 min. Scale bar, 20 μm. Data, means ± SE (n = 5), one-way ANOVA with Tukey’s multiple comparisons. Analysis of GLUT1 puncta/cell (F, G) and its localization to endolysosomal compartments (% of LAMP1 profiles positive for GLUT1 immunostaining), (H) phenotype monitored by HCM quantification in Huh7^WT cells transfected with GFP (control) or GFP-Rab7^WT, GFP-Rab7^Q67L, and GFP-Rab7^T22N, expressing plasmids. Scale bar, 20 μm. Data, means ± SE (n = 3); one-way ANOVA with Tukey’s multiple comparisons. Analysis of GLUT1 puncta/cell (I, J) by HCM quantification in Huh7 WT and ATG16L1-KO cells complemented with Flag (control), Flag-ATG16L1^FL, or Flag-ATG16L1^E230, expressing plasmids. Scale bar, 20 μm. Data, means ± SE (n = 5); one-way ANOVA with Tukey’s multiple comparisons. HCM images in all relevant panels, examples from a bank of unbiased operator-independent machine-collected and algorithm-processed fields containing a minimum of 500 primary objects/cells per well (5 wells minimum per 96-well plate; 3 plates minimum), per cell line/condition.

Figure 7—figure supplement 1. CASM agonists effects on retromer cargo GLUT1 in the absence of changes in TBC1D5-LC3A association.

(A) High content microscopy (HCM) images of GLUT1 puncta in Huh7^WT cells upon treatment with Monensin (100 µM), LLOMe (100 µM), and Bafilomycin A1 (100 nM) for 45 min. Coimmunoprecipitation (co-IP) analysis (B) and quantification of TBC1D5 (C) and VPS35 (D) interaction with GFP-LC3 in Huh7 cells treated with or without Monensin (100 µM), LLOMe (100 µM), and Bafilomycin A1 (100 nM) for 45 min. Data, means ± SE (n = 3), one-way ANOVA with Tukey’s multiple comparisons. HCM images in A, examples from a bank of unbiased operator-independent machine-collected and algorithm-processed fields containing a minimum of 500 primary objects/cells per well (5 wells minimum per 96-well plate; 3 plates minimum), per condition.