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. 2021 Feb 19;220(4):e202010048. doi: 10.1083/jcb.202010048

Figure 1.

Figure 1.

Starvation inhibits endocytic recycling of multiple transmembrane proteins, and autophagy deficiency abolishes the inhibition. (A) Confocal microscopy imaging for GLUT1 (gray) and LAMP2 (red) immunofluorescence staining in HeLa cells stably expressing GFP-LC3 (green). The cells were starved for 4 h in EBSS in the presence or absence of 100 nM BafA1 (E+B). Representative images of three independent experiments are shown. Scale bars: 10 µm. Quantification of GLUT1 immunofluorescence on LAMP2-positive lysosomes. Results for individual cells are plotted, along with the mean and SD for each group (n = 77 cells for control [Con], n = 74 for EBSS, and n = 54 for E+B); P values were calculated using one-way ANOVA by Tukey’s HSD test or t test. ****, P < 0.0001. (B) Confocal microscopy imaging of internalized CD8A-SEMA4C (green) and phalloidin/EEA1/LAMP1 (red) immunofluorescence staining in HeLa cells. HeLa cells were transfected with a plasmid encoding CD8A-SEMA4C for 24 h, followed by incubation with anti-human CD8A antibody on ice for 30 min. Unbound antibodies were removed, and the internalization of antibody-bound CD8A was performed in complete medium, or EBSS in the presence or absence of 100 nM BafA1 (E+B) for 0, 1, or 3 h. Representative images of two independent experiments are shown. Scale bars: 10 µm. Pearson’s correlation coefficients for CD8A-SEMA4C and phalloidin/EEA1/LAMP1. Results for individual cells are plotted, along with the mean and SD for each group: CD8A-SEMA4C versus phalloidin (n = 43 cells for control, n = 29 for EBSS, and n = 54 for E+B), CD8A-SEMA4C versus EEA1 (n = 35 cells for control, n = 31 for EBSS, and n = 39 for E+B), and CD8A-SEMA4C versus LAMP1 (n = 45 cells for control, 48 for EBSS, 44 for E+B); P values were calculated using one-way ANOVA by Tukey’s HSD test or t test. ****, P < 0.0001;***, P < 0.001; *, P < 0.05. (C) Confocal microscopy imaging of internalized CD8A-PTHR (green) and phalloidin/EEA1/LAMP1 (red) immunofluorescence staining in HeLa cells. Cells were transfected with a plasmid encoding CD8A-PTHR and treated as in B. Representative images of two independent experiments are shown. Scale bars: 10 µm. Pearson’s correlation coefficients for CD8A-PTHR and phalloidin/EEA1/LAMP1. Results for individual cells are plotted, along with the mean and SD for each group: CD8A-PTHR versus phalloidin (n = 41 cells for control, n = 33 for EBSS, and n = 47 for E+B), CD8A-PTHR versus EEA1 (n = 59 cells for control, n = 86 for EBSS, and n = 89 for E+B), and CD8A-PTHR versus LAMP1 (n = 46 cells for control, n = 43 for EBSS, and n = 60 for E+B); P values were calculated using one-way ANOVA by Tukey’s HSD test or t test. ****, P < 0.0001; ***, P < 0.001; **, P < 0.01. (D) Starvation results in specific loss of GLUT1 and SEMA4C at the cell surface. HeLa cells were starved for 4 h in EBSS in the presence or absence of 100 nM BafA1 (E+B), followed by surface biotinylation. One of three independent experiments is shown. Representative immunoblots show the protein levels in the surface (PM) and total fractions (lysis). (E) Confocal imaging for GLUT1 (green) and LAMP2 (red) immunofluorescence staining of WT and ATG7-KO HEK293T cells. The cells were starved for 4 h in EBSS in the presence or absence of 100 nM BafA1 (E+B). Representative images from three independent experiments are shown. Scale bars: 10 µm. Pearson’s correlation coefficients for GLUT1 and LAMP2. Results for individual cells are plotted, along with the mean and SD for each group (293T-WT control, n = 37 cells; 293T-WT EBSS, n = 72 cells; 293T-WT E+B, n = 71 cells; 293T-ATG7-KO control, n = 63 cells; 293T-ATG7-KO EBSS, n = 59 cells; and 293T-ATG7-KO E+B, n = 45 cells); P values were calculated using one-way ANOVA by Tukey’s HSD test or t test. ****, P < 0.0001; **, P < 0.01. HSD, honestly significant difference; ns, not significant.