## Results
### Trafficking of N-Terminally Truncated Human Cathepsin B-EGFP in HCT116 Cells
Pronounced co-localization was further observed for the chimeric protein with patchy Golgi 97 staining in close proximity to the nucleus (Figure 5C), while co-localization of the chimeras with Lamp2-containing vesicles of the endocytic pathway was observed as well (Figure 5D). These results demonstrated the expected trafficking route followed by CB-EGFP in HCT116 cells, i.e., its biosynthesis at the rER followed by transport to the Golgi apparatus and targeting to the final destination of cathepsin B in endo-lysosomes. The results also provided evidence for intact trafficking pathways taken by hydrolytic enzymes to the endocytic compartments of CRC cells, and highlighted that EGFP-tagging itself did not affect sorting, folding, and trafficking of cathepsin B in HCT116 cells. Expression of the 52 NCB-EGFP chimeras in HCT116 cells resulted in the formation of profound juxtanuclear localized structures of considerable size (Figure 5E, arrow) in addition to cytosolic distribution. Occasionally, green signals were observed in the nuclei of HCT116 cells, possibly indicative of the nuclear localization of 52 NCB-EGFP. However, the nuclear compartments of HCT116 cells expressing 52 NCB-EGFP did not show any  
Figure 5: Trafficking of full-length and N-terminally truncated human cathepsin B in HCT116 cells. Micrographs of HCT116 cells after transfection with phCB-EGFP (**A–D**) and ph52 NCB-EGFP (**E–H**). Merged (**A–H**) single channel fluorescence (right panels, top and middle) and corresponding phase contrast micrographs (right panels, bottom) were taken with a confocal laser scanning microscope. Immunolabeling with anti-cathepsin B antibodies revealed co-localization with the expressed full-length chimeric protein (**A**, yellow signal) but not the N-terminally truncated cathepsin B form (**E**). To detect the sub-cellular localization of the expressed chimeric proteins in detail, compartment-specific antibodies were used, such as PDI for the ER (**B**,**F**), Golgi 97 for the Golgi apparatus (**C**,**G**) and Lamp2 for endo-lysosomes (**D**,**H**). Note that chimeric full-length cathepsin B followed the expected trafficking pathway, indicated by yellow signals derived from co-localization with compartment-specific markers. In contrast, the N-terminally truncated cathepsin B showed prominent accumulation in structures adjacent to the nuclei of HCT116 cells (**E**, arrow), and did not show co-localization with any compartment-specific marker for the secretory and endocytic pathways (**F**,**G**,**H**), absence of yellow signals). Nuclei were counter-stained with Draq5. Scale bars represent 20 \(\upmu\)m.  
yellow signal for co-localization with the red signals indicative of anti-cathepsin B-stained structures (Figure 5E), thereby most probably indicating cytosolic cleavage of the chimeric protein and sorting of EGFP to the nuclei of HCT116 cells. No co-localization was detected for 52 NCB-EGFP with any of the compartment-specific markers (Figure 5F-H, arrows). 52 NCB-EGFP was typically confined to irregularly shaped focal structures in the juxtanuclear region of HCT116 cells (Figure 5E-H, arrows); however, these did not co-localize with the endo-lysosomal marker protein Lamp2. The results suggested that N-terminally truncated human cathepsin B is possibly synthesized at ribosomes in the cytoplasm and trafficked differently than full-length cathepsin B in HCT116 cells. In this cell line, 52 NCB-EGFP accumulated in juxtanuclear foci that resembled aggresomes. These cytoplasmic structures, in which mis-folded proteins are stored away, are typically found in juxtanuclear position close to the microtubule-organizing center (MTOC) [41].