Figure 5. Canonical autophagy does not affect sorting of the retromer cargo GLUT1.

(A, B) High content microscopy (HCM) quantification of GLUT1 (immunostaining of endogenous protein) puncta/cell in in Huh7^WT, Huh7^ATG13-KO, Huh7^FIP200-KO, Huh7^ATG5-KO, and Huh7^VPS35-KO cells. Scale bar, 20 μm. Data, means ± SE (n = 5); one-way ANOVA with Tukey’s multiple comparisons. (C, D) HCM quantification of GLUT1 (immunostaining of endogenous protein) colocalization with LAMP2 (% of LAMP2 profiles positive for GLUT1; overlap area) in in Huh7^WT, Huh7^ATG13-KO, Huh7^FIP200-KO, Huh7^ATG5-KO, and Huh7^VPS35-KO cells. Scale bar, 20 μm. Data, means ± SE (n = 5); one-way ANOVA with Tukey’s multiple comparisons. (E–G) HCM quantification of GLUT1 (immunostaining of endogenous protein) puncta/cell (E) and GLUT1 colocalization with LAMP2 (% of LAMP2 profiles positive for GLUT1; overlap area) (F) in Huh7^WT, Huh7^FIP200-KO, Huh7^{FIP200-KO + siATG5}, and Huh7^VPS35-KO cells. Scale bar, 20 μm. Data, means ± SE (n = 6); 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.