Detection of extracellular and intracellular HMGB1 protein in CLL samples. (A) HMGB1 and (B) DNA concentrations were determined in the plasma of healthy (n = 14) and CLL samples (untreated, n = 22; relapsed, n = 7; partial remission, n = 7). Significantly increased HMGB1 and DNA concentrations in CLL samples were expressed as median ± interquartile range and differences analyzed by Student t test. (C) Correlation between HMGB1 and DNA concentration in 36 cases of CLL plasma. Dots in the box are 5 cases with the highest concentrations of both HMGB1 and DNA and represent the group of patients with the poorest prognosis. (D-E) Correlations between plasma (D) HMGB1 or (E) DNA concentration with ALC. ALC information was collected at the time of blood draw (supplemental Table 3). (F-H) Determination of HMGB1 expression in CLL and normal B cells by western blotting. (G) Representative results from 2 cases of CLL and 4 normal B-cell samples. (H) HMGB1 expression in long-term cryopreserved CLL samples. Fifty micrograms of proteins was loaded into each lane. Mouse anti-HMGB1 antibody (Sigma-Aldrich) was used at 1:3000 dilution. β-actin was used as a loading control. Numbers below each pair of bands are ratios of HMGB1 to β-actin. (I) Detection of HMGB1 intracellular localization in freshly isolated CLL cells by immunofluorescent staining. DAPI indicates nuclear localization. The isotype control images are demonstrated in supplemental Figure 1A-B.