Effect of the expression of Cdc42N17 on superoxide production.

Kinetics of superoxide production are shown on the left side of panels A and B. Transfected HL-60 cells were grown and differentiated into neutrophil-like cells with Bt₂cAMP (A) or DMSO (B) in the absence or presence of doxycycline (50 ng/mL). Superoxide formation was initiated by the addition of fMLFK or PMA (arrow), and cytochrome c reduction was continuously recorded at 550 nm. Data are representative of more than 10 independent experiments. On the right side of panels A and B, superoxide production was measured in differentiated cells grown in intermediate doses of doxycycline. Results are expressed as percentage of the response obtained in uninduced cells. Values represent the means ± SD of 3 independent experiments. An immunoblot representative of the dose-dependent expression ofmyc-Cdc42N17 is presented. (C) Oxidase activity assay in a cell-free system. Membrane and cytosolic fractions from induced (black bars) and uninduced (gray bars) Bt₂cAMP-differentiated HL-60 cells (dHL-60) were assayed for their ability to stimulate superoxide production in an amphiphile-activated heterologous cell-free system. Results are expressed as the percentage of the response obtained with cytosol of doxycycline-treated cells and bovine membrane or with membrane of doxycycline-treated cells and bovine cytosol. Values represent the means ± SD of 5 independent experiments performed in duplicate. (D) [³⁵S]GTPγS loading of Rac. [³⁵S]GTPγS was added to the cell-free system in the preincubation step. GTPγS-loaded Rac was pulled down with a GST fusion of the PAK1 Rac-binding domain. The figure is representative of 3 independent experiments.