Figure 8.

Regulation of the chemotactic and transmigration responses of lymphocytes by silencing of HDAC6. (A) HDAC6-GFP or HDAC6 H216A/H611A-GFP overexpression enhanced the migratory ability of PBLs versus GFP overexpression. Nucleofected PBLs overexpressing HDAC6-GFP, HDAC6 H216A/H611A (HDAC6 DD), or GFP were allowed to migrate toward a chemotactic gradient of 100 ng/ml SDF-1α in Transwell chambers. Two independent experiments with different healthy donors are shown. (B) Effect of HDAC6 knocking-down in PBL chemotaxis. Western blotting analysis of HDAC6 and acetylated tubulin in siRNA HDAC6-interfered PBLs. Two independent migration assays in Transwell chambers, with HDAC6 knocked-down PBLs versus mock nucleofected with control negative oligonucleotides from different healthy donors, are shown. (C) Rescue of defective migratory activity of HDAC6 knockdown cells. CEM cells were interfered with siRNA HDAC6, and 48 h later were transfected with either wild type or DD (double deacetylase dead mutant) and then assayed for chemotactic migration in Transwell chambers. (D) HDAC6 knockdown inhibits lymphocyte transendothelial migration under flow. Rolling, adhesion, transmigration, and detachment events were quantified for negative control siRNA (dark gray bars) and HDAC6 siRNA (white bars) treated cells under physiological shear stress on a TNF-α–activated HUVEC monolayer. The number of cells in each step was quantified as described in Material and Methods. Control Western blotting analysis of HDAC6 and alpha-tubulin in siRNA HDAC6-interfered CEM cells is shown. Data show mean values ± SD from four independent experiments.