Figure 3: Microvillar localization of GLUT1 and facilitation of glucose transport in FSS-exposed BeWo cells.

Cells were seeded in the chamber area of the device and then cultured overnight under static or fluid flow (2 μl min⁻¹) conditions. (a,b) Localization of GLUT1 (green) was analysed by immunofluorescence confocal microscopy. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Representative x–y optical sections at apical or basal side of cells and the stacked images were shown. Scale bar, 20 μm. (c) Representative confocal images of x–z optical sections of BeWo cells stained with GLUT1 antibody (green) and phalloidin (F-actin, magenta). The lowest panel (Flow+Cyto D) shows the image derived from cells cultured under fluid flow conditions in the presence of cytochalasin D (Cyto D, 3 μM). Scale bar, 10 μm. (d) Relative mRNA expression of SLC2A1 (GLUT1) in BeWo cells cultured with or without FSS. BeWo cells were cultured overnight under static or fluid flow (2 μl min⁻¹) conditions, and SLC2A1 mRNA expression normalized to 18S ribosomal RNA level was analysed by real-time PCR. The data are presented as the mean±s.e.m. from three independent experiments. Data significance was assessed by unpaired two-tailed Student’s t-test; *P<0.05. (e) The subcellular localization of GLUT1 was analysed by immunoelectron microscopy. GLUT1 signals (arrowheads) were detected at the microvillar surface of the FSS-exposed cells. The boxed area is magnified in e′. Scale bars, 3 μm (e); 1 μm (e′). (f,g) Facilitation of glucose uptake and transfer to the fetal channel in the FSS-exposed cells. The glucose transport activity of BeWo cells was analysed by 2-NBDG (2 mM) uptake assays. The data are presented as the mean±s.d. (n=5 for each condition). Significance was assessed by unpaired two-tailed Student’s t-test; *P<0.05.