Effect of PLD1 gene expression knock-down on cell migration. (A) cDNA map of PLD1b showing the exons to which 2 duplex siRNAs target. (B) PLD1-siRNAs and a negative control siRNA were transfected into dHL-60 cells, RNA was isolated and used to analyze gene expression by QRT-PCR using a FAM-labeled PLD1 probe multiplexed with a Texas-Red-labeled housekeeping gene. (C) Gene fold expression was calculated from ΔCt as indicated in “Materials and methods” (error bars are SEM with n = 3 in duplicate). Duplicate cell samples were used to generate cell lysates for the detection of protein expression by Western blotting. Blots were cut in half, the upper part (containing an ∼ 110-kDa region) was probed with anti-PLD1 antibodies, whereas a lower part (containing an ∼ 45-kDa region) was probed with anti-β-actin, to ascertain equivalent protein loading. (D) Analysis of chemokinesis/chemotaxis. Cells were resuspended in RPMI-based chemotaxis buffer at a 5 × 10⁵ cells/mL density and placed on the upper or insert chambers of 6.5-mm Transwell plates. Appropriate bottom wells contained either buffer only, 30 nM ENA-78, 50 nM FMLP, or 12 nM IL-8; 100% represents 1.3 × 10⁴ ± 0.15 cells/mL migrating to the bottom wells.