Fig. 5: REEP1 vesicles do not colocalize with known cellular compartments.

a U2OS cells stably expressing REEP1-mCh were transfected with the early endosomal marker GFP-Rab5, grown in complete DMEM media, and imaged by confocal microscopy. Right panel shows the overlay between REEP1-mCh (green) and GFP-Rab5 (magenta). Scale bar, 2 µm. b As in (a), but with transfection of the late endosomal marker GFP-Rab7 (magenta). c As in (a), but with transfection of the recycling endosomal marker GFP-Rab11 (magenta). d As in (a), but analyzed by immunostaining with anti-ATG9A antibodies (αATG9A, magenta). Top row shows a cell grown in DMEM and the bottom row a cell starved in EBSS for 30 min. e Pearson’s correlation analysis of REEP1-mCh or -mEm colocalization with various autophagy or vesicular markers in U2OS cells cultured in full or starvation medium (− or + 30 min EBSS). See text for a description of markers. Note that all Pearson’s values are under 0.5, indicating that REEP1 signals do not extensively correlate with any marker tested. Shown are means and standard deviations. P-values were calculated using one-way ANOVA analysis, multiple comparisons (Sidak’s method), ****p < 0.0001, ns, not significant. The cell number (n) analyzed for each condition is listed above each data set. Exact p-values are listed in the Source Data. f As in (a), but in cells coexpressing Listeria phospholipase C and the C1 tandem domains of protein kinase D fused to mEm, to visualize PIPEROsomes (magenta). g As in (a), but in cells stably expressing REEP1-mEm (green) and stained with the neutral lipid dye Lipidtox (magenta) to visualize lipid droplets. h As in (g), but in cells transfected with mCh-PIS (magenta), which localizes to both the ER and ER-associated punctae. i As in (g), but in cells immunostained with anti-Sec31 antibodies (αSec31, magenta) to visualize ER exit sites. j As in (f), but in cells immunostained with anti-PMP70 (αPMP70, magenta) to visualize peroxisomes.