HGAL induces RhoA activation. (A) Raji and VAL lymphoma cells, transfected with siRNA for HGAL or scrambled control siRNA 48 hours before the experiments, and HeLa cells stably transfected with pcDNA3.1-HGAL or pcDNA3.1-mock plasmids, were starved for 8 hours and then seeded on fibronectin for 60 minutes. Cellular extracts were prepared and RhoA, CDC42 and Rac1 pull down assays were performed. HGAL knockdown and equal loading were confirmed by immunoblotting with HGAL and actin antibodies. Results are representative of 3 independent experiments. Densitometry analysis of normalized RhoA-GTP to total RhoA is presented. The values in the control samples were arbitrarily defined as 1. Error bars represent SD; * indicates a statistically significant difference (P < .05) between experimental conditions. Normalized densitometry measurements for CDC42 and HGAL are also shown below the corresponding blots. (B) RhoA pull down assays in CD77+ GC B cells and CD77– cells enriched from normal tonsils. (C) B lymphocytes, enriched from peripheral blood of healthy volunteers, were transfected with control or HGAL-encoding plasmids. 48 hours later the cells were stimulated with LPA and RhoA activity was assessed in triplicates by G-Lisa RhoA Activation Assay kit (Cytoskeleton Inc). * indicates a statistically significant difference (P = 3.3 × 10−5). Fraction of the same lymphocytes was used in IL-6 chemotaxis assay, demonstrating significant (P = 6.6 × 10−4) inhibition in chemotaxis of HGAL-expressing normal lymphocytes.
