Rituximab induces A-SMase and CER localization in the outer leaflet of the cell membrane. (A) Cells (3 × 10⁶) were treated with or without 10 μg/mL rituximab for 10 minutes and analyzed by confocal microscopy by using rabbit anti-SMase and/or mouse antihuman Fc. Results are representative of 3 independent experiments. (B) Rituximab induces outer leaflet A-SMase localization in B-CLL fresh cells. Intact cells (3 × 10⁶) from patient 1 were treated or not with 10 μg/mL rituximab for 10 minutes and analyzed by flow cytometry using a FITC-labeled rabbit polyclonal anti-A-SMase. Results are representative of 3 independent experiments. (C) Intact cells (3 × 10⁶) were treated (green line) or not (blue line) with 10 μg/mL rituximab for 10 minutes and analyzed by flow cytometry by using a FITC-labeled mouse monoclonal anti-CER 15B4. Results are representative of 3 independent experiments. A stained FITC anti-mouse served as control (black line). (D) Rituximab induces outer leaflet CER accumulation in B-CLL fresh cells. Intact cells (3 × 10⁶) from patient 1 were treated or not with 10 μg/mL rituximab and analyzed by flow cytometry by using a FITC-labeled mouse monoclonal anti-CER 15B4. Results are representative of 3 independent experiments. (E) Bacterial SMase induces outer leaflet CER accumulation in Daudi cells. Intact cells (3 × 10⁶) were treated or not with 0.2 U bacterial SMase and analyzed by flow cytometry by using a FITC-labeled mouse monoclonal anti-CER 15B4. Results are representative of 3 independent experiments. (F) C₁₆-CER mimics rituximab-induced inhibitory cell viability. Daudi cells were treated or not (○) with C₁₆-CER at 0.05 μM(•), 0.1 μM(▴), or 0.5 μM(▪), and cell viability was estimated by trypan blue exclusion assay. Results are the mean of 3 independent experiments ± SD.