Figure 1

Phenotypic differences in autophagic flux and endocytosis in VPS KO cells. (a) LC3 flux in VPS KO HeLa-Kyoto cells. WT and VPS KO cells were cultured in the absence or presence of BafA1 for 2 h. Total cell extracts were analysed for LC3 and β-actin (ACTB) by western blotting. Representative blots are shown from 3 independent experiments. (b) Quantitative analysis of band intensities in panel (a). Intensities of LC3 divided by ACTB were normalized to BafA1-treated WT cell samples as 1. Autophagic flux shown in the right panel was calculated as follows: (LC3^BafA1+–LC3^BafA1−)/LC3^BafA1+. Mean and S.D. are shown. Overlayed-circle represents each data point. Statistics were calculated by one-way ANOVA followed by post hoc Tukey’s test. (c) Receptor-mediated endocytosis in VPS KO HeLa-Kyoto cells. WT and VPS KO cells were stimulated with 100 ng/mL of EGF for 2 h in the absence or presence of BafA1. The whole cell lysates were analysed for EGFR and ACTB by western blotting. Representative blots are shown from 3 independent experiments. (d) Quantitative analysis of band intensities in panel (c). Mean and S.D. from 3 independent experiments are shown. EGFR degradation was determined by the following formula: (EGFR^BafA1+–EGFR^BafA1−)/EGFR^BafA1+. Overlayed-circle represents each data point. Statistics were calculated by one-way ANOVA followed by post hoc Tukey’s test. (e) DQ Red-labelled BSA was incorporated into WT and VPS KO HeLa-Kyoto cells for 4 h and fluorescent images of activated DQ Red were acquired by confocal microscopy. A representative image from each sample is shown. Bars = 10 µm. (f) Quantitative analysis of DQ Red-BSA intensity in panel (e). Five images for each cell were captured, and analysed by Cell Profiler™. Integrated fluorescence intensities of DQ Red for each cell are normalized to the median value of WT and are displayed as a box-and-whisker plot. The whisker shows either the range of the data points or 1.5 IQR at maximum. Each circle represents the outlier that is outside of the whisker range. The Boxes represent the upper quartile (top line), median (middle bar), and lower quartile (bottom line), respectively. The counted cells for WT; n = 57, VPS41 KO; n = 40, VPS8 KO; n = 44, VPS39 KO; n = 50, VPS3 KO; n = 56. Statistics were calculated by the Kruskal–Wallis test followed by the Mann–Whitney U test with Holm correction. * p < 0.05, ** p < 0.01, *** p < 0.001, and n.s.; not significant.