FIGURE 3.

iPLA₂ knockdown inhibits super-oxide generation in neutrophils A, HL60 cells (1 × 10⁶ /ml) were differentiated with 1.25% DMSO in RPMI 1640 media supplemented with 10% HIFBS and D-glucose (5.5 mM) for 3 d. On day 3, cells were transfected with nontargeting siRNA (1 μM) and si-iPLA₂ (1 μM) by nucleofection and re-suspended in the differentiation media. Nontransfected differentiated cells served as controls. After 72 h, cells were collected, and RNA was extracted by Trizol reagent and reverse transcribed to cDNA. Real-time PCR using probes for actin and iPLA₂ was set to 45 cycles to estimate the fold change of iPLA₂ normalized to actin. B, Figure shows a typical amplicon of real-time PCR. C, Cells transfected with si-iPLA₂ and nontargeting siRNA were counted and resuspended in PBS at a concentration of 25 × 10⁶/ml and stimulated with 1 μM fMLP. Cytochrome C reduction was read in a spectrophotometer at 550 nm for triplicate samples. Data are presented as nanomoles of O₂⁻ and expressed as mean ± SEM. p < 0.05.