Fig. 4

Arginine depletion-mediated decrease in cell adhesion and motility is through the stimulation of autophagy in ovarian cancer cells. a Left panel: Micrographs illustrating SKOV3 cells treated with hArgI or rapamycin and stained with Cyto-ID (green). Fluorescent images were taken using the 63x objective lens of the Zeiss Observer Z1 fluorescent microscope. Scale bar is 10 μm. b LC3 cleavage in SKOV3 (upper panel) and Caov-3 (lower panel) and actin blot for loading control (lower gels) following treatment with hArgI or rapamycin on western blot. c SKOV3 cells were treated with hArgI, rapamycin or hArgI in combination with chloroquine before being imaged moving randomly in media. Cell images were captured at 1 min interval for 2 h.The average migrated distance (in μm) was measured by tracking the images using ImageJ software and the speed was calculated by dividing the distance over time. The bar graph illustrates the fold change in migration of hArgI, rapamycin or hArgI + chloroquine treated SKOV3 cells normalized to the untreated control. d Representative micrographs of fixed and stained control SKOV3 cells, and SKOV3 cells treated with either hArgI, rapamycin or hArgI in combination with chloroquine. Scale bar is 100 μm. Right panel: Colorimetric quantification of the dissolved crystal violet stain of the cells described in C 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 untreated control. Significance was set at p < 0.05. e Bar graph illustrating the number of focal adhesions in control and hArgI or rapamycin treated cells stained with anti-vinculin. The number of focal adhesion was quantified using the CLAHE and Log3D plugins in Image and is expressed as absolute values of the means for every sample