Scale bars: 10?m. lymphoma and primary B-cell malignancies from patients with rituximab-refractory, suggesting the potential clinical application to combat rituximab resistance. < .001 for each compared with the PBS control), 11B8 could significantly prolong the survival of SCID/Raji-R mice (Fig.?1H). More importantly, treatment with the cathepsin inhibitor E-64d could markedly decrease the protection of 11B8 in both SCID/Raji and SCID/Raji-R mice, while the significant difference was not observed in the rituximab-treated groups. De novo synthesis of ceramide is essential for LMP-mediated cell death initiated by type II CD20 mAb Ceramide, a prototypic sphingolipid, is either synthesized de novo or generated from sphingomyelin breakdown.26 To evaluate the notion that ceramide is involved in the LMP-mediated cell death induced by type II CD20 mAbs, widely used specific inhibitors of A-SMase (Imipramine), N-SMase (3-O-Methyl-sphingomyeline) and ceramide synthase (fumonisin B1) were employed. 11B8-induced cell death can be significantly inhibited by 25?M fumonisin B1 (FB1), whereas imipramine (Imip) and 3-O-Methyl-sphingomyeline (3-OMe-SM) could not protect the cells (the concentration from 50?M to 0?M)(Fig.?2A). The FB1, in the range from 0.2?nM to 25?nM, exhibited a dose-dependent inhibition of cell death induced by 11B8 (Fig.?S2A). Exogenous ceramide (either C2 ceramide or ceramide obtained from bovine spinal cord) could induce dose-dependent cell death in both Raji and Ramos cells in the concentrations from 150?M to 50?M (Fig.?2B). After treatment with 10?g/mL CD20 mAbs, the generation of ceramide induced by 11B8 was detectable after 6?h (Fig.?2C). The elevation of intracellular ceramide stimulated by 11B8 could be specifically inhibited by FB1 (Fig.?2D). The generation of Ceramide and the inhibitory effect of FB1 were also confirmed by the confocal fluorescent microscopy analysis (Fig.?S2B). Moreover, LMP and the subsequent release of cathepsin B into the cytosol could be markedly inhibited by FB1 (Fig.?2ECG). Treatment with exogenous ceramide (150?M or 100?M) could significantly induce LMP and the subsequent ACR 16 hydrochloride ACR 16 hydrochloride release of cathepsin B (Fig.?2E and H). Open in a separate window Figure 2. ceramide synthesis involved in LMP-mediated cell DHX16 death initiated by type II CD20 mAb. (A) The inhibition of cell death in Ramos cells by A-SMase, N-SMase and ceramide synthase inhibitors (Imip, 3-OMe-SM and FB1, respectively) was assessed by FCM. Error bars indicate SD (n = 3). *p < 0.05. (B) The exogenous ceramide (C2-Ceramide) induced cell death in both Raji and Ramos cells in a dose-dependent manner. *p < 0.05. (C) Time course study of ceramide generation in B cells induced by CD20 mAbs. The ceramide levels were quantitated as described in Supplemental Experimental Procedures. Results are representative of three independent experiments. (D) The generation of ceramide stimulated by 11B8 was inhibited by FB1. Raji cells were treated with FB1 prior to the addition of CD20 mAbs. *p < 0.05. Detection of total lysosomal volume in cells treated with mAbs and FB1. Cells were incubated with CD20 mAbs (10?g/mL) and FB1 (25?M). After that, cells were labeled with LysoTracker green and the volume ACR 16 hydrochloride of the lysosomal compartment measured by confocal microscopy (E) and FCM (F) after 4?h. (G and H) The assessment of LMP by evaluating the release of cathepsin B (red) into cytoplasm. Scale bars: 10?m. Dihydroceramide desaturase-1 (DEGS1) is critical to the initiation of LMP-mediated cell death DEGS1, a key enzyme in the de novo pathway of ceramide generation, is the only dihydroceramide desaturase reported to be present in human cells.27 Here, shRNA against the human desaturase enzyme DEGS1 was used to attenuate its expression and consequently inhibit its activity. Raji and Ramos cells were transfected with DEGS1 shRNA or nonspecific shRNA. Knockdown of DEGS1 mRNA level was confirmed by quantitative reverse transcriptase PCR (qRT-PCR), with about 75% knockdown of DEGS1 mRNA achieved in Raji and Ramos cells (Fig.?S3A). The decreased expressions of the DEGS1 protein in DEGS1 shRNA-treated Raji (Raji/DEGS1-) and Ramos (Ramos/DEGS1-) ACR 16 hydrochloride cells were also confirmed by protein gel blotting (Fig.?S3B and C). The decrease of DEGS1 expression and consequent reduction of its activity could effectively prevent the cell death induced by 11B8 in both Raji and Ramos cells (Fig.?3A and B). The cathepsin.