Figure 4: FSS induces Ca²⁺ entry and increase of intracellular [Ca²⁺] in BeWo cells.

(a) Representative Ca²⁺ responses in BeWo cells at each indicated time point. Pseudocolored images are shown: red cells indicate high levels of intracellular [Ca²⁺] measured by the fluorescence intensity, and blue cells represent basal levels. Scale bar, 50 μm. (b) Time course of Fura-2 fluorescence in the BeWo cells. FSS was transiently loaded by infusing the medium at flow rates of 5 (t=2.0–4.2 min), 0.5 (t=10.9–13.6) and 2 (t=18.7–21.8 min) μm min⁻¹. The calcium response images (a) were captured at the time points indicated by arrows, respectively (t=0, 5, 23 min). Data are shown as the Fura-2 ratio (F₃₄₀/F₃₈₀) and mean±s.d. (n=24). (c–f) Inhibition of FSS-induced microvilli formation by calcium chelators. SEM images of BeWo cells cultured under fluid flow (5 μm min⁻¹) in the presence or absence of EGTA (1 mM) or BAPTA-AM (10 μM) for 3 h. The representative images (c–e) were captured at the centre area of the chamber, and total length of microvilli per field (700 μm², five fields) was measured (f). The data represent the mean±s.d.; **P<0.01, ***P<0.001, Student’s t-test. Scale bar, 5 μm.