BAG3 Protein Is Involved in Endothelial Cell Response to Phenethyl Isothiocyanate

Figure 1

PEITC induces actin cytoskeleton alterations and activation of the PI3K/Akt pathway in HUVECs. (a) HUVECs from 2 different donors were plated at 5 ×10³/cm² and then treated with PEITC at indicated concentrations for 24 and 48 h. Then, cells were subjected to the MTT assay, and results are shown in a bar graph as % of viable cells in respect to controls (untreated or 0.01% DMSO-treated cells). (b) HUVECs from 2 different donors were treated as described above. After 24 h, a cell cycle assay was performed by using PI on permeabilized cells. Results are expressed as % of cells in each cell cycle phase. (c) Phase-contrast images and confocal analysis of HUVECs treated with (A, D) DMSO (control) at a final concentration (0.01%), with (B) PEITC (10 μM) for 15 min, and with (C, E) PEITC (10 μM) for 120 min. (B, C, E) Images of PEITC-treated cells showing blebbing formation in the outer cytoskeletal domain. (d) HUVECs were seeded as described above and treated with PEITC; at the indicated time points, cells were harvested and subjected to cell lysis. Protein contents were analyzed by Western blot to analyze the levels of phospho-FAK protein. Tubulin was used as a loading control. (e) HUVECs were treated with 0.01% DMSO (C) and with 10 μM PEITC for 15, 30, 60, and 120 min. Total protein extracts were analyzed by Western blot using anti-phospho-Akt (Ser473), anti-Akt, anti-phospho-PI3K, and anti-PI3K to test PI3K/Akt activation levels. The anti-GAPDH antibody was used as an internal loading control. Each lane was loaded with 20 μg protein. The bar graph depicts densitometric analysis of the data (expressed as phospho-PI3K/PI3K and phospho-Akt/Akt ratios) corresponding to the left panel. Results were obtained from at least two independent experiments and are expressed as the mean ± SEM. , , and , statistically significant differences, compared to DMSO-treated cells (C), were calculated by Student’s t-test for unpaired data.

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