Fig. 1: Deregulation of the SIRT3/SOD2 axis in CLL cells influences ROS generation.
A, B CLL cells contain lower levels of O2− but higher levels of H2O2. Purified CLL cells or normal B-cells were stained with DHE to detect O2− levels (A) or DCFDA to detect accumulation of H2O2 (B) and analyzed by flow cytometry. Results are presented as mean fluorescent intensity (MFI) ± one standard deviation. C SOD2 remains as constitutively active. Mitochondrial fractions isolated from purified CLL cells or normal B-cells (N1–N4) (obtained from panels A/B) were analyzed for acetylated-SOD2 (Ac-SOD2 [K68]) in western blot using a specific antibody. Total mitochondrial SOD2 was used as loading control. Densitometric analysis of the blots was also performed to determine relative levels of Ac-SOD2 (right panel). D CLL cells overexpress SIRT3. Purified normal B-cells from healthy subjects (N5–N9) or CLL cells (P2, P3, P10–P16) were analyzed for the expression of SIRT3 in western blot using a specific antibody. Actin was used as loading control. Densitometric analysis was performed to show relative change. E ROS inhibition reduces SIRT3 level. Purified CLL cells (P57, P63, P64) treated with a ROS-inhibitor Trolox (50 µM/4 h) or left untreated were analyzed for SIRT3 expression as in panel D. GAPDH was used as loading control. Densitometric analysis was performed to show relative change (lower panel). F ROS activates SIRT3 expression. Purified CLL cells (P59, P63) treated in vitro with H2O2 (50 µM and 100 µM) for 4 h were analyzed for SIRT3 expression as described in panel E. Densitometric analysis was performed to show relative fold-increase of SIRT3. G BCR activation induces SIRT3 expression. Purified CLL cells (P65, P68) treated with an anti-IgM antibody for the indicated time periods were analyzed for the expression of SIRT3 and P-BTK levels (as an indicator of BCR activation) in western blots using specific antibodies. BTK and GAPDH were used as loading controls.
