Figure 1.
Expression and localization of ANXA1 in human skin fibroblast cells innormal and high glucose conditions.
(A) Intracellular (Cytosol), cell surface (EDTA wash) and extracellular (Supernatants) ANXA1 expression from WS1 cells after 3 days treatment in LG and in HG conditions, was analyzed by Western blot with anti-ANXA1 antibody. The optical density of the protein bands was normalized on tubulin levels. The relative intensities of bands were determined using Gel-Doc 2000 software (BIO-RAD) and to the control band was given an arbitrary value of 100. ***p<0.001 vs LG. (B) Cultured human WS1 fibroblasts fixed and labeled with fluorescent antibody against ANXA1 (red) and with FITC-conjugated Phalloidin (green) in LG (a, b, c) and HG conditions (d, e, f). In normal glucose conditions ANXA1 protein is highly expressed in WS1 fibroblast cells and accumulates at the plasma membrane and at lamellipodial extrusions (arrows). An increase of glucose levels leads to a reduction of ANXA1 expression and inhibits the relocation of the protein to the plasma membrane (e). The data are representative of 5 experiments with similar results. Bar = 25 µm.
Figure 2.
Effects of ANXA1 knock down or neutralization on WS1 cell migration.
(A) Cell extracts from WS1 cells treated or not with siRNAs direct against ANXA1 in LG or HG conditions were analyzed by Western blot using an anti-ANXA1 antibody. The optical density of the protein bands was normalized on tubulin levels. ***<0,001, **<0,01 vs LG; °°°<0,001, °°<0,01 vs HG. (B) Cell migration into a scraped wound of WS1 cells treated or not with siRNAs against ANXA1 in LG or HG conditions. Results are reported as means of three experiments, measuring individual cell migrations 24 h after scraping. Bars represent standard errors. ***<0.001 vs LG; °°<0.01 vs HG. (C) Cell migration into a scraped wound of WS1 cells treated or not with fMLP (50 nM), Ac2-26 (1 µM) and an ANXA1 blocking antibody (1∶100) in LG or HG conditions. Results are reported as means of three experiments, measuring individual cell migrations 24 h after scraping. Bars represent standard errors. ***<0.001 vs LG; °°°<0.001, °°<0.01 vs HG.
Figure 3.
FPR expression and activation in WS1 fibroblasts.
(A) Cell surface expression of FPR-1 and FPR-2 in human WS1 fibroblasts was analyzed by flow cytometry. FPR1 (green) is highly express in WS1 cells whereas a weak expression of FPR-2 (purple) is observed. (B) Effects of Ac2-26 (1 µM), fMLP (50 nM) and CsH (500 nM) on the FPR-induced rise in intracellular Ca2+. WS1 were treated as described in Materials and Methods. The histogram shows the fluorescence ratio calculated as F340/F380 nm in the presence or in the absence of extracellular Ca2+. Control represents unstimulated cells. Data are means ± SEM (n = 3). *** <0.001, ** <0.01 vs corresponding controls; °°° <0.001, °° <0.01 vs corresponding Ac2-26 or fMLP. (C) ERK phosphorylation rate was analyzed by Western blot at different time point following Ac2-26, fMLP or CsH incubation. The Western blot is representative of the 20 minutes experimental time point. The protein levels were normalized on the α-tubulin expression. The data are representative of 3 experiments with similar results. *** <0.001; ** <0.01 vs control.
Figure 4.
Ac2-26 stimulates direct migration of WS1 in normal and high glucose conditions.
Results for control, fMLP (50 nM), Ac2-26 (1 µM), CsH (500 nM), fMLP + CsH and Ac2-26+ CsH are reported as means of three experiments, measuring individual cell migrations 24 h after scraping. Bars represent standard errors; ***<0.001 vs LG; °°°<0.001 vs HG.
Figure 5.
Hyperglycemia induces a slight increase of FPR1 expression in WS1 Cells.
(A) Cell surface expression of FPR-1 in human WS1 fibroblasts incubated in LG or HG conditions was analyzed by flow cytometry. FPR1 expression is slightly increased in WS1 cells treated for 3 days in HG conditions (green) compared to LG conditions (violet). Data are representative of 3 experiments with similar results. (B) Cell surface expression of FPR-1 in human WS1 fibroblasts treated (green) or not (violet) with Ac2-26 peptide was analyzed by flow cytometry. The obtained results show that the Ac2-26 peptide is not able to induce any modulation of FPR1 expression. Data are representative of 3 experiments with similar results.