Figure S1.

Starvation inhibits endocytic recycling of multiple transmembrane proteins, and autophagy deficiency abolishes the inhibition. (A) Confocal microscopy imaging for GLUT1 (gray) and β-catenin (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 β-catenin-positive cellular membranes. Results for individual cells are plotted, along with the mean and SD for each group (n = 63 cells for control, n = 65 for EBSS, and n = 85 for E+B); P values were calculated using one-way ANOVA by Tukey’s HSD test or t test. ****, P < 0.0001. (B) Confocal imaging for TRAILR1 (gray) and phalloidin (red) immunofluorescence staining in HeLa cells stably expressing GFP-LC3 (green). The cells were treated as in A. Representative images of three independent experiments are shown. Scale bars: 10 µm. Pearson’s correlation coefficients for TRAILR1 and phalloidin. Results for individual cells are plotted, along with the mean and SD for each group (n = 88 cells for control, n = 85 for EBSS, and n = 108 for E+B); P values were calculated using one-way ANOVA by Tukey’s HSD test or t test. (C) Confocal imaging for GLUT1 (green) and phalloidin (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 phalloidin. Results for individual cells are plotted, along with the mean and SD for each group (293T-WT con, n = 45 cells; 293T-WT E+B, n = 28; 293T-ATG7-KO con, n = 34; 293T-ATG7-KO E+B, n = 36); P values were calculated using one-way ANOVA by Tukey’s HSD test or t test. ****, P < 0.0001. (D) Confocal imaging for GLUT1 (green) and LAMP2 (red) immunofluorescence staining of WT and ATG7 KO MEF cells. The cells were starved for 4 h in EBSS in the presence or absence of 100 nM BafA1 (E+B). Representative images 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 (MEF-WT con, n = 10; MEF-WT E+B, n = 19; MEF-ATG7-KO con, n = 19; MEF-ATG7-KO E+B, n = 19); P values were calculated using one-way ANOVA by Tukey’s HSD test or t test. ****, P < 0.0001. (E) Confocal imaging for Venus-GLUT1 (green) and LAMP2 (red) immunofluorescence staining of WT and ATG7 KO HEK293T cells. The cells were transfected with a plasmid encoding Venus-GLUT, and starved for 4 h in EBSS in the presence or absence of 100 nM BafA1 (E+B). Representative images from two independent experiments are shown. Scale bars: 10 µm. Pearson’s correlation coefficients for Venus-GLUT1 and LAMP2. Results for individual cells are plotted, along with the mean and SD for each group (293T-WT con, n = 35; 293T-WT EBSS, n = 34; 293T-WT E+B, n = 32; 293T-ATG7-KO con, n = 27; 293T-ATG7-KO EBSS, n = 47; 293T-ATG7-KO E+B, n = 40); P values were calculated using one-way ANOVA by Tukey’s HSD test or t test. ****, P < 0.0001. HSD, honestly significant difference; ns, not significant.