## Results
### Cathepsin B of HCT116 Cells during Cell Cycle Progression  
Further studies were performed to analyze the possible effects of cathepsin B forms on cell cycle progression of HCT116 cells in analogy with our previous investigations of the role of the related cysteine cathepsins L in this cell line [35]. Localization of cathepsin B was studied in HCT116 cells in different phases of the cell cycle. In particular, HCT116 cell cultures were synchronized and arrested in the G1/G0, S, and G2/M phases of the cell cycle by serum starvation for 24 h, a double thymidine block, and incubation with nocodazole, respectively. This protocol for preparation and the purity of sub-cellular fractions of synchronized and cell cycle-arrested HCT116 cells was previously proven [32; 35]. Immunofluorescence labeling with anti-cathepsin B antibodies showed vesicles confined to the peri-nuclear region of HCT116 cells arrested in the G1/G0 phase (Figure 7A). HCT116 cells arrested in the S phase also exhibited cathepsin B prominently in vesicles which were more abundant than in the G1/G0 phase (Figure 7C, cf. Figure 7A). During the G2/M phase, however, cathepsin B-positive vesicles were dispersed throughout the cytoplasm, and cathepsin B staining was also detected in association with condensed chromosomes (Figure 7E, yellow signals). Immunoblotting of whole cell lysates (W), lysosomal (Ly) and nuclear fractions (Nu) of HCT116 cells upon arrest in different cell cycle stages, respectively, was performed to study the expression of the different molecular forms of cathepsin B (Figure 7B,D,F, respectively). During the G1/G0 phase, cathepsin B was predominantly present as inactive proform (Figure 7B, pro), while the mature forms of cathepsin B were mainly detected in the nuclear fractions (Figure 7B, Nu, SC, and HC). Fully processed forms of cathepsin B, i.e., mature single- and two-chain forms, were present in all fractions prepared from HCT116 cells arrested in the S-phase, and peaked in the lysosomal fraction (Figure 7D, Ly). Interestingly, the 40-kDa specific form of cathepsin B was observed in both the nuclear and lysosomal fractions of S phase-arrested HCT116 cells (Figure 7D, arrowhead). In cells arrested in the G2/M phase, a band representing proacthepsin B was most prominent in the nuclear fraction of HCT 116 cells (Figure 7F, Nu), while mature cathepsin B was detectable only in the lysosomal fraction (Figure 7F, Ly). The activity analysis of cathepsin B in these sub-cellular fractions showed highest lysosomal activities during the S phase (Figure 7H), whereas nuclear cathepsin B activities reached a maximum in the G1/G0 phase, i.e., before cells entered the S phase. The results indicated a possible involvement of cysteine cathepsin B activity in facilitating S phase entry of HCT116 cells, which was approached subsequently. Figure 6: N-terminally truncated human cathepsin B forms are present in aggresomes of HCT116 cells. Aggresome formation in HCT116 cells inspected at 24 h post-transfection with ph52 NCB-EGFP, as detected by using the ProteoSata® aggresome detection dye. Confocal laser scanning micrographs demonstrating merged (**–D**,**A**_**  
To study whether the enzymatic activity of cathepsin B was required at all for cell cycle progression of HCT116 cells, sub-confluent cultures of HCT116 cells were serum-starved for 24 h for synchronization in G1/G0 phase and treated with the cathepsin B-specific inhibitor CA-074 throughout the chase period. No effect of cathepsin B inhibition was observed in such cell cultures (Figure 8A). It is important to note that neither the distribution of cathepsin B-inhibited nor that of CB-EGFP-expressing HCT116 cells in the different cell cycle phases was altered to the distribution across cell cycle phases in non-treated HCT116 cell cultures (Figure 8 cf. [35]). Figure 7: Cathepsin B expression pattern during the cell cycle of HCT116 cells.