Fig. 5.

Knockdown of PI3K p110δ with siRNA attenuates eosinophil rolling, adhesion, activation-induced shape change, and migration. A: expression of PI3K p110δ in BM-Eos after transfection with scrambled siRNA or PI3K p110δ siRNA by Western blot analysis using polyclonal antibodies against PI3K p110δ (1:250, top) along with densitometry (bottom). B: expression level of PI3K p110δ mRNA in BM-Eos after transfection with scrambled siRNA or PI3K p110δ siRNA detected by qPCR. Expression levels of β-actin served as the internal control. C: rolling of BM-Eos transfected with scrambled siRNA or PI3K p110δ siRNA on rm VCAM-1-coated coverslips under conditions of flow in vitro. Rolling of BM-Eos treated with DMSO (vehicle for IC87114) is shown for comparison. D: adhesion of fluorescently labeled BM-Eos after transfection with scrambled siRNA or PI3K p110δ siRNA to immobilized rm VCAM-1 or rm ICAM-1 as described in Fig. 1. Adhesion of untreated BM-Eos is shown as a control. E: confocal microscopy of FITC-phalloidin-stained BM-Eos transfected with scrambled siRNA or PI3K p110δ siRNA adhered to rm VCAM-1 (magnification ×600). F: migration of BM-Eos transfected with scrambled siRNA or PI3K p110δ siRNA toward 100 nM murine eotaxin-1 in Transwell plates after 2 h at 37°C. Data in A (top) and E are representative of 2–4 independent experiments. Combined data (mean ± SE) of 3–4 independent experiments are shown for A (bottom), B, C, D, and F. *P < 0.01 in B, D, and F and < 0.03 in A (bottom) and C for comparison of scrambled siRNA vs. PI3K p110δ siRNA-transfected eosinophils.