Figure 1.

PGE2 alters proliferation, migration, survival and adhesion of pericytes. (a) Human brain microvascular pericytes (HBVPs) were cultured for 72 h with increasing concentrations of PGE2 and a wound closure assay was performed to assess their migration activity. The areas of uncovered surface were determined in 9 micrographs from 3 different dishes using ImageJ software. Representative data from three independent experiments performed in triplicates are shown. (b) PGE2-treated HBVPs were seeded in Transwell inserts for migration assay. Eight hours later, the migrating cells were fixed and stained. The stained area was measured in 12 micrographs from 4 Transwells for each condition. A representative result of two independent experiments performed in quadruplicates is shown. (c) HBVPs were incubated with various concentrations of PGE2 for 72 h, detached from the cell culture dish by 0.005% trypsin and seeded in 24-well plates for 30 to 45 min in basal medium, at 37 ºC. Unattached cells were removed by PBS wash, and cells were fixed and stained with methanol/crystal violet solution. Crystal violet was subsequently extracted from attached cells to measure the absorbance at 595 nm. The experiment is representative of three experiments performed in quadruplicates. (d) HBVPs were treated with either control DMSO, PGE2 10 nM or PGE2 100 nM for 72 h before protein extraction. Western blot analysis was performed to assess the expression of proteins involved in cell adhesion: Src/phospho-Src, FAK/phospho-FAK and paxillin/phospho-paxillin. GAPDH was used as an internal control. One-way ANOVA, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant.