Blocking differentiation of NLCs. (A,C,E) CLL cells were cultured for 1 week individually with different neutralizing antibodies or inhibitors. Suspending CLL cells were removed gently prior to adding the MTT solution. After incubation with MTT for 2 hours, a large amount of formazan was formed inside NLCs. Images of NLCs were taken under phase-contrast microscopy, which are displayed as larger dark cells on the bottom of plates. (B,D,F) To quantify the amount of NLCs, formazan in the cells was dissolved with isopropanol, and OD values were measured by spectrophotometry. Reduced numbers of NLCs were expressed as percentage of control. Data shown are mean ± standard deviation from 3 individual cases CLL patients with triplicate OD values. Significant changes were analyzed by the Student t test. (A-B) Blocking HMGB1, RAGE, or TLR9. Fresh CLL cells (5 × 106/mL) in 24-well plates were incubated with 10 µg/mL of anti-HMGB1 or anti-RAGE neutralizing antibody or 100 nM G-iODN for 1 week. (C-D) Inhibition of HMGB1 by EP. Cells were treated with 1.0, 2.5, and 5.0 mM of EP for 1 week. (D) Viability of CLL cells were determined by a Vi-Cell XR Cell Viability Analyzer, and MTT data represent the amount of NLCs before and after treatment with EP. (E-F) Blockade of RAGE, IL-6, or TLR4 by neutralizing antibodies. CLL cells were incubated with 10 µg/mL of anti-RAGE, anti–IL-6, or TLR4 neutralizing antibody for 1 week.
