Fig. 3

Arginine depletion decreases cell adhesion by inhibiting RhoA in SKOV3 cells. a Control and hArgI or hArgI + citrulline treated SKOV3 cell extracts were incubated with GST-RBD beads before blotting against RhoA. Total cell lysates which were not incubated with the beads were also blotted against RhoA and used as loading control. Right panel: Bar graph illustrating the amount of RhoA activation expressed as fold change of the activated RhoA normalized to the amount of total protein. The densitometry quantification of active and total RhoA bands was performed in ImageJ. b SKOV3 ovarian cancer cells were transfected with Luciferase siRNA or RhoA siRNA for 72 h before protein extraction and blotting against RhoA. Left panel: Western blot profile showing the expression levels of RhoA and beta actin in SKOV3 cells. Right panel: Quantification of RhoA expression levels by ImageJ. The data is presented as fold decrease in RhoA expression levels normalized to beta actin of the siRNA RhoA transfected cells relative to the luciferase control. Data are the mean ± SEM from 3 assays. The results were significant with p < 0.05. c SKOV3 cells were transfected with Luciferase siRNA, RhoA siRNA, or treated with hArgI and transfected with an empty vector or a constitutively active RhoA construct, respectively. All samples were grown to confluency before creating a wound in the cell monolayer and imaging the wound area at (t = 0) and 72 h later (t = 72). The micrographs represent the wound profile of the same area at the indicated time points. Scale bar is 100 μm. Right panel: Bar graph illustrating the quantification of the wound closure rate expressed as fold changes relative to the luciferase control. The results were significant with p < 0.05. d Left panel: Representative micrographs of fixed and stained SKOV3 cells which were transfected with Luciferase siRNA, RhoA siRNA, or treated with hArgI and transfected with an empty vector or the constitutively active RhoA construct, respectively. Scale bar is 100 μm. Right panel: Colorimetric quantification of the dissolved crystal violet stain was performed at 560 nm using an Elisa plate reader. The bar graph represents the quantification of adherent cells expressed as fold increase relative to the luciferase control. Significance was set at p < 0.05