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. 2025 Jan 7;13:RP100928. doi: 10.7554/eLife.100928

Figure 4. Membrane atg8ylation apparatus affects sorting of the retromer cargo GLUT1.

(A, B) High content microscopy (HCM) quantification of GLUT1 (endogenous protein immunostaining) puncta/cell in Huh7WT, Huh7ATG3-KO, Huh7ATG5-KO, Huh7ATG7-KO, Huh7ATG16L1-KO, and Huh7VPS35-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 (endogenous protein immunostaining) colocalization with LAMP2 (% of LAMP2 profiles positive for GLUT1; overlap area) in Huh7WT, Huh7ATG3-KO, Huh7ATG5-KO, Huh7ATG7-KO, Huh7ATG16L1-KO, and Huh7VPS35-KO cells. Scale bar, 20 μm. Data, means ± SE (n = 5); one-way ANOVA with Tukey’s multiple comparisons. (E, F) HCM quantification of GLUT1 (endogenous protein immunostaining) colocalization with LAMP2 (% of LAMP2 profiles positive for GLUT1; overlap area) in HeLaWT, and HeLaHexa-KO cells. 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.

Figure 4—source data 1. Numerical values for quantification in graphs.

Figure 4.

Figure 4—figure supplement 1. ATG5 knockout has no effect on protein levels of retromer subunits.

Figure 4—figure supplement 1.

Immunoblot analysis (A) and quantification (B) of retromer complex proteins VPS35 (i), VPS26 (ii), and VPS29 (iii) from Huh7WT, Huh7ATG3-KO, Huh7ATG5-KO, and Huh7ATG7-KO cells (total cell extract). Immunoblot analysis (C) and quantification (D) of retromer complex proteins VPS35 (i), VPS26 (ii), and VPS29 (iii) from HeLaWT, and HeLaATG5-KO cells (total cell extract). (E) High content microscopy (HCM) quantification of GLUT1-LAMP2 colocalization in HeLaLC3-TKO, HeLaGABA-TKO, and HeLaHEXA-KO cells. Data, means ± SE (n = 4), one-way ANOVA with Tukey’s multiple comparisons; ns (not significant), p > 0.05. (F) Immunoblot analysis showing the siRNA-mediated knockdown of ATG5 in Huh7FIP200-KO cells. (G) HCM images of GLUT1 puncta/cell in Huh7WT, Huh7FIP200-KO, Huh7FIP200-KO+ siATG5, and Huh7VPS35-KO cells. HCM images in panel F, 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 4—figure supplement 1—source data 1. PDF files containing original immunoblots for Figure 4—figure supplement 1 indicating relevant bands.
Figure 4—figure supplement 1—source data 2. Original files for immunoblots displayed in Figure 4—figure supplement 1.
Figure 4—figure supplement 1—source data 3. Numerical values for quantification in graphs.