Figure 2. Rituximab induced HMGB1 release.
A-C. Four DLBCL cell lines in 2 × 106/ml were treated with rituximab for 4 hours. HMGB1 in the conditioned medium (50 μl) and cytosolic extracts were determined by Western blotting using a mouse anti-HMGB1 antibody. BSA and β-tubulin were used as loading controls for the conditioned medium and the cytosolic proteins, respectively. (A) A group of representative Western blots; (B and C) Statistical analysis of HMGB1 protein levels in the conditioned medium (B) and cytosolic extracts (C) Data shown were mean ± SD from 4 DLBCL cell lines. ‘***’ in (B) indicates significant difference of HMGB1 levels in the conditioned medium between R-CHOP and CHOP treated samples. D. HMGB1 translocation. Cells were treated with rituximab alone for 4 hours. Fixed/Permeabilized cells were co-stained with HMGB1 antibody showing red and DAPI (blue) for nuclear localization. E. GA-101 antibody-induced HMGB1 release. Cells were incubated with or without 10 μg/ml GA-101 antibody for 4 hours. HMGB1 in the conditioned medium was determined by Western blotting. F. Anti-T cell antibody does not induce HMGB1 release in malignant B-cells. Su-6 and Su-8 cell lines were treated with 10 μg/ml anti-CD3 (OKT3) antibody for 4 hours. Rituximab (R)-treated cells were used as a positive control. (G and H) Comparison of the patterns between HMGB1 and cathepsin D release. G. DoHH2 cells were treated with rituximab for 4 hours. Fixed/Permeabilized cells were co-stained with anti-HMGB1 (red) and anti-cathepsin D (green) antibodies, and DAPI (blue). Arrow shows the colocalization of HMGB1 and cathepsin D. The single color images were shown in the Supplemental Figure 2A. H. Colocalization analysis. PDM images and correlation coefficient (Rr) were generated by the intensive correlation analysis using ImageJ software. The orange color and higher Rr indicate a colocalization and the blue color and a lower or negative Rr means a segregation.