Fig. 1. (A) CaSR stimulation generates PM ruffling. HEK cells were transfected with SEP-CaSR. 48 hours post-transfection, cells were challenged (bottom) or not (top) with 5 mM CaCl₂ for 10 minutes, fixed and stained for SEP-CaSR (green) and F-actin (Alexa-Fluor-568–phalloidin, red). Colocalisation areas appear in yellow in the merge panels. Cells exhibiting PM ruffles are shown by empty arrowheads while cells that did not change shape are indicated by filled arrowheads. Bars, 10 µm. (B) Representative micrographs of CaSR-agonist-induced cytoskeleton reorganisation in SEP-CaSR-expressing HEK cells before (untreated) and after 5 mM CaCl₂ or 10 µM NPS R-467 stimulation for 10 minutes. Colocalisation of SEP-CaSR (green) and F-actin (red) appears in yellow in the merge panels. The right panels show fourfold magnified views of the boxed regions. Note the presence of SEP-CaSR at the edge of protrusions. The PM ruffles are shown by arrowheads. Bars, 5 µm. Pictures shown are representative of six independent experiments. (C) Quantification of the effects of CaSR agonists on PM ruffling of cells. These results are the mean±s.e.m. of three to six independent experiments. ^*P0.001 compared with controls. (D) Extracellular-calcium-mediated PM ruffling is CaSR-dependent. Representative confocal images of F-actin staining of wild-type (WT) or stably expressing CaSR HEK cells challenged with 5 mM CaCl₂ for 10 minutes, in the presence (+) or absence (–) of CaSR antagonist (NPS 89636 at 1 µM, pre-incubated for 10 minutes). Ruffling cells are indicated by arrowheads. Bars: 10 µm. (E) Quantification of cells exhibiting PM ruffling in the conditions described in (D). The results shown are the mean± s.e.m. of three independent experiments. ^*P0.001 compared with untreated. (F) Time course of CaSR-induced PM ruffling. The percentage of SEP-CaSR-expressing cells exhibiting PM ruffling after different times of CaCl₂ stimulation was determined. All time points of stimulation were significantly different (P0.001) from the untreated (time 0 minutes) condition.