Figure 6. Membrane atg8ylation machinery is required for proper RAB7 localization.

(A–C) High content microscopy (HCM) quantification of Rab7 (endogenous protein immunostaining) puncta/cell (B) and Rab7 colocalization with LAMP2 (% of LAMP2 profiles positive for GLUT1; overlap area) (C) in Huh7^WT, Huh7^ATG3-KO, Huh7^ATG5-KO, Huh7^ATG7-KO, Huh7^ATG16L1-KO, and Huh7^VPS35-KO cells. Scale bar, 20 μm. Data, means ± SE (n = 6); one-way ANOVA with Tukey’s multiple comparisons. (D, E) Immunoblot analysis and quantification of proteins in lysosomes purified/enriched by LysoIP (immunoisolation with TMEM192-3xHA) from Huh7^WT, Huh7^ATG5-KO, and Huh7^VPS35-KO cells. TMEM192-2xFLAG, negative control. Data, means ± SE (n = 3), one-way ANOVA with Tukey’s multiple comparisons. (F–H) HCM quantification of Rab7 (endogenous protein immunostaining) puncta/cell (G) and Rab7 colocalization with LAMP2 (% of LAMP2 profiles positive for GLUT1; overlap area) (H) 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 = 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.

Figure 6—figure supplement 1. Loss of membrane atg8ylation but not of canonical autophagy diverts of RAB7 to lysosomal compartments.

(A) High content microscopy (HCM) images of Rab7 (immunostaining of endogenous protein) showing Rab7 colocalization with LAMP2 (% of LAMP2 profiles positive for GLUT1; overlap area) in Huh7^WT, Huh7^ATG3-KO, Huh7^ATG5-KO, Huh7^ATG7-KO, Huh7^ATG16L1-KO, and Huh7^VPS35-KO cells. Scale bar, 20 μm. (B) HCM images of Rab7 (puncta/cell of endogenous Rab7 profiles stained for immunofluorescence) in Huh7^WT, Huh7^ATG13-KO, Huh7^FIP200-KO, Huh7^ATG5-KO, and Huh7^VPS35-KO cells. Scale bar, 20 μm. HCM images (C) and quantification of RAB7 puncta (D) in Huh7^WT, Huh7^FIP200-KO, Huh7^{FIP200-KO + siATG5}, and Huh7^VPS35-KO cells. Scale bar, 20 μm. Data, means ± SE (n = 8), one-way ANOVA with Tukey’s multiple comparisons. HCM images (E) and quantification of RAB7-LAMP2 colocalization (F) in Huh7^WT, Huh7^FIP200-KO, Huh7^{FIP200-KO + siATG5}, and Huh7^VPS35-KO cells. Scale bar, 20 μm. Data, means ± SE (n = 8), 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.