*4.9. Cell Growth Determination*

The CL-48 cells were plated in six-well cell culture plates at 20,000 cells/well in 2 mL culture medium containing fetal bovine serum. The cells were treated as indicated and harvested by suspension in 0.025% trypsin containing 0.02% EDTA. Cell counts were performed in triplicate while using a hemocytometer with trypan blue exclusion to identify the viable cells. Growth curves were generated.

Cell cycle analysis of CL-48 cells was carried out by quantifying the DNA content with propidium iodide staining and using a FACScan instrument with CellQuest software (Becton Dickinson).

#### *4.10. Filamentous Actin (F-actin) Fluorescence Staining*

HUVECs were cultured on cover slides and, after reaching confluence, the HUVECs were treated with CM. After 1 h, HUVECs were washed with serum-free M199 medium, fixed with 3.7% paraformaldehyde for 20 min, and permeabilized with 0.1% Triton-X-100. Fluorescence isothiocyanate-conjugated phalloidin (Invitrogen, Carlsbad, CA, USA), diluted in PBS (2 U/mL), was then applied to the specimens in the dark for 1 h. The specimens were mounted with 10% glycerol and images were acquired using a fluorescence microscope (Nikon, Tokyo, Japan).

## *4.11. HUVEC Monolayer Permeability Assays*

HUVECs were cultured in Transwell chambers (0.4 μm pore polycarbonate filters; Costar, Cambridge, MA, USA). After reaching confluence, the medium was replaced with the CM (0.3 mL in the upper chamber and 1 mL in the lower chamber). Horseradish peroxidase molecule (Sigma-Aldrich, Saint Louis, MO, USA) was added to the upper compartment at a concentration of 0.2 μM. After incubation for 1 h, the medium in the lower compartment was assayed for enzymatic activity while using a photometric guaiacol substrate assay (Sigma-Aldrich).
