Figure 3
Figure 3. NADPH-oxidase activity and antioxidant machinery in CLL cells. (A) Relative gene expression of the NOX subunits is shown comparatively for purified HD B-cells (n = 15) and CLL cells (n = 22). Differences of the relative gene expression of gp91 were validated using FACS and determining the gp91 MFI of normal B-cells (n = 5) and CLL cells (n = 5). (B) Superoxide anions generated by the membrane-bound NOX were quantified based on cytochrome c reduction in purified HD B-cells (n = 4) and CLL cells (n = 5) at baseline conditions (unstim), on addition of the NOX-substrate NADPH, and after direct NOX activation using PMA. (C) Intracellular ROS levels are shown (left panel) as the CellROX-MFI in purified HD B-cells (n = 10) and CLL cells (n = 8) with and without the addition of NOX-activating PMA. The mean fold-increase of CellROX-MFI in B-cells and CLL cells on NOX activation is displayed (right panel). (D) The relative gene expression of key cellular antioxidants (catalase, cat; manganese superoxide dismutase, mnsod; heme-oxygenase-1, HO1; thioredoxin-1, trx1) is shown comparatively for purified HD B-cells (n = 10) and CLL cells (n = 22) as quantified by qPCR. (E) Cellular glutathione (GSH) levels were detected in HD B-cells (n = 6) and CLL cells (n = 6) using the Thioltracker probe and were semiquantified based on the Thioltracker-MFI by FACS. (F) The relative gene expression of key molecules involved in GSH-synthesis and (G) the regeneration of reduced GSH were evaluated in purified HD B-cells (n = 8-10) and CLL cells (n = 10-17). (H) Mean intracellular levels of the important cellular reducing molecule NADPH that is produced within the pentose phosphate pathway are shown for purified HD B-cells (n = 3) and CLL cells (n = 4) as measured by ELISA. (I) The overall antioxidant capacity was measured in lysates from normal B-cells (n = 7) and CLL cells (n = 9). Bars indicate the standard error mean. Abbreviations: P, P-value; *P < .05; **P < .005; ***P < .001. GCLC, catalytic subunit of the glutamate-cysteine ligase; GCLM, modifier subunit of the glutamate-cysteine ligase; G6PDH, glucose-6-phosphate dehydrogenase.

NADPH-oxidase activity and antioxidant machinery in CLL cells. (A) Relative gene expression of the NOX subunits is shown comparatively for purified HD B-cells (n = 15) and CLL cells (n = 22). Differences of the relative gene expression of gp91 were validated using FACS and determining the gp91 MFI of normal B-cells (n = 5) and CLL cells (n = 5). (B) Superoxide anions generated by the membrane-bound NOX were quantified based on cytochrome c reduction in purified HD B-cells (n = 4) and CLL cells (n = 5) at baseline conditions (unstim), on addition of the NOX-substrate NADPH, and after direct NOX activation using PMA. (C) Intracellular ROS levels are shown (left panel) as the CellROX-MFI in purified HD B-cells (n = 10) and CLL cells (n = 8) with and without the addition of NOX-activating PMA. The mean fold-increase of CellROX-MFI in B-cells and CLL cells on NOX activation is displayed (right panel). (D) The relative gene expression of key cellular antioxidants (catalase, cat; manganese superoxide dismutase, mnsod; heme-oxygenase-1, HO1; thioredoxin-1, trx1) is shown comparatively for purified HD B-cells (n = 10) and CLL cells (n = 22) as quantified by qPCR. (E) Cellular glutathione (GSH) levels were detected in HD B-cells (n = 6) and CLL cells (n = 6) using the Thioltracker probe and were semiquantified based on the Thioltracker-MFI by FACS. (F) The relative gene expression of key molecules involved in GSH-synthesis and (G) the regeneration of reduced GSH were evaluated in purified HD B-cells (n = 8-10) and CLL cells (n = 10-17). (H) Mean intracellular levels of the important cellular reducing molecule NADPH that is produced within the pentose phosphate pathway are shown for purified HD B-cells (n = 3) and CLL cells (n = 4) as measured by ELISA. (I) The overall antioxidant capacity was measured in lysates from normal B-cells (n = 7) and CLL cells (n = 9). Bars indicate the standard error mean. Abbreviations: P, P-value; *P < .05; **P < .005; ***P < .001. GCLC, catalytic subunit of the glutamate-cysteine ligase; GCLM, modifier subunit of the glutamate-cysteine ligase; G6PDH, glucose-6-phosphate dehydrogenase.

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