Figure 5. Baf plus Cu causes ATP7B to accumulate in large EEA1-positive endosomes located predominantly at the peripheral region of WIF-B cells.

Cells were treated same as Figure 4 but fixed at 15 minute intervals during the 60 minutes of Cu-treatment plus DMSO or 50 nM Baf, then double-stained for ATP7B (green) and EEA1 (red). Substantial overlap between A) ATP7B and B) EEA1 was seen in large clusters at the peripheral region of cells as early as 15 minutes after Cu plus Baf. C) Magnification of a group of typical Baf-induced clusters (each ∼5 μm across), with ATP7B concentrated in subdomains (arrows) of the large and more homogeneous EEA1-positive endosomes. D) 60 minutes after Cu plus DMSO or E) Cu plus Baf. Single confocal planes from the D-E) nuclear or D'-E') peripheral region show overlap between ATP7B and EEA1 persisted in Baf-treated cells. 3D renderings of confocal image stacks representing 0, 15 and 60- minute time points are shown in Supplementary Movie S4. F) The extent of overlap between ATP7B and EEA1 during 60 minutes of Cu plus Baf was quantified from at least 4 confocal stacks from a single experiment. The 0 and 60 minute time points represent the mean of 10 confocal stacks, obtained from 3 independent experiments. G) To characterize the effect of Baf on the EEA1-positive endosomes, the mean cross-sectional area of the large EEA1 structures was measured across all experimental conditions and plotted against the time that the cells were incubated in Baf or DMSO. H) The fraction of total EEA1 that was present in “large” EEA1 organelles, defined as having > 1 μm² cross-sectional area in the 2D analysis.