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Galectin-9 is the most potent inducer of cell death in lymphomas and other malignant cell types among the members of the galectin family. We investigated the mechanism of galectin-9-induced cell death in PC-3 prostate cancer cells in comparison with in Jurkat T cells. Galectin-9 induced apoptotic cell death in Jurkat cells, as typically revealed by DNA ladder formation. On the other hand, DNA ladder formation and other features of apoptosis were not apparent in PC-3 cells undergoing galectin-9-induced death. Exogenous galectin-9 was endocytosed and destined to the lysosomal compartment in PC-3 cells. The internalized galectin-9 was resistant to detergent solubilization but was solubilized with lactose. Agents inhibiting actin filament dynamics abolished the internalization and cytocidal effect of galectin-9 in PC-3 but not Jurkat cells. Galectin-9 induced accumulation of ubiquitinated proteins, possibly heterogeneously ubiquitinated and/or monoubiquitinated proteins, in PC-3 cells. PYR-41, an inhibitor of the ubiquitin-activating E1 enzyme, suppressed the cytocidal effect of galectin-9. Although ubiquitination was upregulated also in Jurkat cells by galectin-9, PYR-41 was ineffective against galectin-9-induced cell death. Colocalization of ubiquitinated proteins and LAMP-1 was detectable in PC-3 cells treated with galectin-9. The ubiquitinated proteins were recovered in the insoluble fraction upon cell fractionation. In contrast, ubiquitinated proteins that accumulated after treatment with proteasome inhibitors did not co-localize with LAMP-1 and were mainly recovered in soluble fraction. The results suggest that atypical ubiquitination and accumulation of ubiquitinated proteins in lysosomes play a pivotal role in galectin-9-induced non-apoptotic death in PC-3 cells.