Fig 2: CPAP overexpression causes the formation of MVB-like structures. (A) HEK293T cells were transfected with control plasmid, or GFP-CPAP or myc-CPAP expression vectors for 24 h as described for Fig. 1, fixed with paraformaldehyde and processed for TEM, and representative images are shown in upper panels. Numbers of MVB-like structures per unit cell [GFP n = 13; GFP-CPAP n = 43] and diameters of individual MVB like structures [GFP n = 24; GFP-CPAP n = 161] were calculated from three separate experiments for control and GFP-CPAP vector transfected cells and the Mean ± SD values (lower panels) are shown. p-value is by chi2 test comparing the ratios of MVBs with < 1000 nm and > 1000 nm sizes or ratios of cells/images with < 5 and > 5 MVBs between control and GFP-CPAP cells. Note: MVB number and size were determined in transfected cell preparations containing a mixture of cells with and without GFP expression in an unbiased manner for both groups. (B) HEK293T cells were transfected with GFP-CPAP expression vector (doxy-inducible) for 24 h, treated with doxy to induce protein expression for an additional 24 h, subjected to IF staining for EEA1 (early endosome), CD63 (MVBs), and LAMP1 (lysosomes and late endosome), and images of GFP + and GFP- cells were acquired using Airyscan unit of Zeiss 880 microscope. Maximum projection images of Z-stacks (left panel) and single Z planes (right panel) of relevant representative images from at least 3 independent experiments are shown. (C) HeLa cells were stained for endogenous CPAP along with EEA1, CD63 or LAMP1 and imaged using Airyscan unit of Zeiss 880. Maximum Z-projection images (left panel) and single z planes (right panel) of representative images from 3 independent experiments are shown. White arrows show examples of co-localization. Yellow arrows indicate centriole structures. Please note that brightness of red channel was enhanced to visualize low levels of CPAP staining. Supplemental Fig. 4 shows non-enhanced images. Airyscan IF images were initially processed by Zen software (version 2.0) of Zeiss (https://www.zeiss.com/microscopy/us/products/microscope-software/zen.html), and the final maximum projection and single Z-plane images were generated using ImageJ software version 1.53 (https://imagej.nih.gov/ij/). All results are representative of at least three experiments that produced similar trend in outcomes or include cumulative values from three independent experiments. GraphPad Prism software version 9 (https://www.graphpad.com/scientific-software/prism/) was used for determining p-values.