Fig. 3: VAMP7 mediates transport between ER and CD63+ late endosomes.

A, B WT, VAMP7KO and ATG5KO NRK cells were either left untreated or treated with BafA1 (100 nM) for 60 minutes and stained with Mitotracker and CD63. Scale bar = 10 µm. Pearson’s Correlation Coefficient (PCC) was measured using ImageJ for the co-occurrence of Mitotracker and CD63. One-way ANOVA with Bonferroni’s post hoc test was used to determine the statistical significance. Data is derived from 40 cells from three independent experiments and is presented as the mean ± SEM.****p-val < 0.001, ns non-significant. C, D WT, VAMP7KO and ATG5KO NRK cells were either left untreated or treated with BafA1 (100 nM) for 60 minutes and stained with RTN3 and CD63. Scale bar = 10 µm. PCC was measured using ImageJ for the co-occurrence of RTN3 and CD63. One-way ANOVA with Bonferroni’s post hoc test was used to determine the statistical significance. Data is derived from 40 cells from three independent experiments and is presented as the mean ± SEM. ****p-val < 0.001, ns non-significant. E–H WT, VAMP7KO and ATG5KO NRK cells were either left untreated or treated with BafA1 (100 nM) for 60 min and PLA was performed to quantify the RTN3/CD63 or the VDAC/CD63 interaction. One-way ANOVA with Bonferroni’s post hoc test was used to determine the statistical significance. Data is derived from 50 cells from three independent experiments and is presented as the mean ± SEM. ns non-significant, *p-val <0.05, ** p-val < 0.01, ***p-val < 0.001. Scale bar = 10 µm. I WT NRK cells were either left untreated or treated with BafA1 (100 nM) for 60 minutes and immunostained for RTN3 and CD63. They were imaged using the Leica SP8 Confocal Microscope in the stimulated emission depletion (STED) module to achieve a resolution of about 50 nm. The inset shows ‘annular’ structures of CD63 with RTN3 spots indicated by white arrowheads in close apposition of the CD63 membrane. Scale bar = 5 µm, 1 µm.