FIGURE 2.

Decreased SIRT3 expression in CLL and B cell malignancy lines associated with decreased IDH2 activity. A, Western blots using mitochondrial extract from CLL primary cells and primary B cells from healthy donors probed for SIRT3, IDH2, and AcK413-IDH2 (the acetylated, less active form of IDH2). This was repeated for a total of 11 CLL samples and 8 normal B cell samples. B, relative SIRT3 expression in primary B cells (n = 8) compared with CLL cells (n = 11). C, Western blots of nuclear and mitochondrial extracts demonstrating the purity of mitochondrial extract, as assessed by the presence of VDAC and the lack of histone H3. D, IDH2 activities were determined from mitochondrial extracts. E, representative histogram of cells stained with DHE for ROS expression. The gray-filled peak represents CLL cells treated with NAC, a negative control, and the black-filled peak represents CLL cells treated with H₂O₂. The mean fluorescence intensity was determined for each sample, and then averages were calculated for all the CLL samples and all the B cell samples. The adjacent bar graph shows average mean channel fluorescence ± S.E. for CLL (n = 5) and normal B cells (n = 5). F, levels of GSH measured in CLL cells and B cells. G, Western blots using mitochondrial extract from the indicated cell lines probed for SIRT3, IDH2, and AcK413-IDH2. H, scatter plot showing the correlation of SIRT3 protein level with acetylated IDH2 level (both from Western blots) in the cell lines (R² = 0.692). I, bar graph showing IDH2 activity in primary B cells compared with the cell lines. J, scatter plot showing the correlation of SIRT3 level (from Western blots) with IDH2 activity (from I) in the cell lines (R² = 0.759). K, correlation of SIRT3 protein expression (from Western blots) with SIRT3 mRNA expression (determined by Q-PCR) for the B cell malignancy lines used in G–J. *, p < 0.05; **, p < 0.01.