Figure 7
CPAP depletion results in defective trafficking of internalized cell surface receptor to MVB/late endosome. (A) HeLa cells expressing control-shRNA or CPAP-shRNA were incubated with Alexa fluor 555-conjugated EGF ligand and left on ice for 1 h. Cells were washed with serum free media and transferred to 37 °C to initiate receptor internalization. Cells were fixed at indicated time-points, permeabilized and stained for CD63 to mark MVB/late endosome. Images were acquired as Z-stacks using Zeiss 880 microscope. Maximum projection images of cells harvested at different time-points (left panel), and single Z-plane of relevant images (upper right panel) and percentage of EGF + vesicular structures that co-localize with CD63 + staining at 60 min time-point (lower right panel) from four independent experiments combined are shown. Vesicular structures of a single Z-plane were counted for each cell area. Supplemental Fig. 9B shows CD63 staining fluorescence intensity of cell areas of multiple experiments. (B) HeLa cells expressing control-shRNA or CPAP-shRNA were treated with EGF for 1 h and subjected to TEM analysis, and representative images (top panel) and mean ± SD values (lower panel) of number of endosome structures (indicated by white arrows) and endo-/lysosomes (electron dense bodies; indicated by yellow arrows) from three independent experiments combined are shown. Vesicular/electron dense structures from 1 μm sections were analyzed between the two groups. All IF images were processed using ImageJ software version 1.53 (https://imagej.nih.gov/ij/). P-value by unpaired t-test. GraphPad Prism software version 9 (https://www.graphpad.com/scientific-software/prism/) was used for determining P-values.
