HGAL induces activation of RhoA downstream effectors regulating actin cytoskeleton. (A-B) Raji and VAL lymphoma cells, transfected with siRNA for HGAL or control siRNA, and HeLa cells stably transfected with pcDNA3.1-HGAL or pcDNA3.1-plasmids, were starved for 8 hours and then seeded on fibronectin for 60 minutes. (A) ROCK enzymatic activity was measured in triplicates in the indicated cells. Data are presented as mean ± SD of the mean. (B) Cellular lysates were immunoblotted for indicated proteins. Densitometry measurements of respective phosphorylated to nonphosphorylated proteins are presented. The values in control specimens were arbitrarily defined as 1. (C) Raji lymphoma cells, transfected with siRNA for HGAL or control siRNA were starved for 8 hours and then seeded on fibronectin for 90 minutes. The cells were stained with anti-HGAL (red), anti-pTyr (green) and DAPI (blue). Interference reflection contrast (IRM) images were obtained and p-Tyr staining intensity was measured and depicted as mean ± SD of the mean. (D) VAL or Raji cells transfected with siRNA for HGAL or control siRNA were starved for 8 hours and then left unstimulated or treated with LPA (1.0 μg/mL) for 45 seconds followed by staining with Alexa-488 phalloidin and analyzed by flow cytomety. (E-F) Serum-starved HeLa cells stably transfected with pcDNA3.1-HGAL or pcDNA3.1-plasmids were seeded on FN-coated slides for 90 minutes. (E) The cells were stained with anti-pTyr (green), anti-paxillin (red) and DAPI (blue); and (F) rhodamine-labeled phalloidin (red); IRM images were obtained and visualized on a Carl Zeiss LSM510/UV confocal microscope. The intensity of each staining was calculated in 25 cell tetrads as described in supplemental Mehtods and depicted as mean ± SD of the mean for p-Tyr and paxillin (E). Results in panels A through E are representative of 3 independent experiments. * indicate statistically significant difference (all below P < .01).