Figure 3.

Inhibition of PDK1 suppressed chemotaxis in neutrophils. Staurosporine suppressed fMLP-induced (A) or IL-8-induced (B) chemotaxis in neutrophils from healthy individuals (n = 5 persons per group). Chemotaxis was measured by the transwell insert method with a 24-well chemotaxis chamber in which a 5-µm-pore-sized filter separates the upper and lower chambers. To investigate staurosporine inhibition on fMLP- or IL-8-induced chemotaxis, we pre-incubated neutrophils with the various indicated concentrations of staurosporine. In the lower chambers, PBS with fMLP (100 nM) or IL-8 (10 nM) was added as chemo-attractant. Neutrophils were allowed to migrate toward the soluble attractants in the lower chambers for 120 min at 37°C in a humidified atmosphere (5% CO₂). Migrating cells were collected and counted by microscopy. Values are mean ± SEM of 3 independent experiments. Asterisk indicates statistically significant compared with vehicle with fMLP or IL-8 (*P < 0.05). PDK1-specific siRNA decreased total PDK1 mRNA and protein expression in HL60 cells. HL60 cells were induced to differentiate (to dHL-60) with DMSO for 3 days to achieve the expression of the neutrophilic phenotype, followed by transfection with PDK1-specific siRNA and non-target siRNA as a control for 48 hrs. HL60 cells were analyzed by Western blotting and real-time RT-PCR. (C) PDK1 siRNA-transfected HL60 cells showed ˜50% reduction in PDK1 mRNA levels compared with vehicle (non-transfected) and non-target siRNA-transfected cells. Values are mean ± SEM of 3 independent experiments. Asterisk indicates statistically significant compared with vehicle (non-transfected) and non-target (*P < 0.05). (D) PDK1 siRNA-transfected HL-60 cells showed a detectable reduction in PDK1 protein levels compared with non-target siRNA-transfected. PDK1 was detected as a single 68-kDa band in differentiated HL60 cells. The viability during the transfection process was not affected (data not shown) compared with non-transfected cells. Results are representative of at least 3 independent experiments. (E) PDK1 siRNA-transfected HL60 cells exhibited decreased fMLP-induced chemotaxis. (F) PDK1 siRNA-transfected HL60 cells exhibited decreased IL-8-induced chemotaxis. Chemotaxis was measured by the transwell insert method with a 24-well chemotaxis chamber in which a 5-µm-pore-sized filter separates the upper and lower chambers. HL60 cells were allowed to migrate toward the soluble attractants in the lower chambers for 120 min at 37°C in a humidified atmosphere (5% CO₂). Migrating cells were collected and counted. Values are mean ± SEM of 3 independent experiments. Asterisk indicates statistically significant compared with non-transfected HL60 cells or transfected with non-target HL60 cells on fMLP- or IL-8-induced chemotaxis (*P < 0.05).