STIM1 and Orai1 regulate invadopodia through Src. (A) Effects of ectopic STIM1 and Orai1 on the levels of pY416 Src, pT308 Akt, and pY397 FAK in WM793 cells. Adherent WM793 cells expressing control vector, STIM1, or STIM1 plus Orai1 were lysed on plates, and 50 µg total cell lysate was loaded onto each lane. Western blotting was performed with respective antibodies as indicated, with GAPDH as a loading control. (B) Effects of ectopic STIM1 and Orai1 on pY416 Src levels in MCF-7 and NMuMG cells; 50 µg total cell lysate was loaded onto each lane. (C) Induction of Ca²⁺ influx with A23187 and thapsigargin (TG) increased Src activity. Adherent WM793 cells were treated with 5 µM A23187 and 2 µM thapsigargin for 30 min before being lysed. (D) Inhibition of SOCE with STIM1 shRNA, 100 µM 2-APB, or 0.5 mM EGTA inhibited Src activity. (E) Scattered dot plot showing that STIM1-mediated invadopodia formation was abrogated by treatment with 2.5 nM dasatinib; n = 51, 50, 51, and 53 for control, STIM1, control + dasatinib, and STIM1 + dasatinib, respectively. (F) Scattered dot plot showing ectopic expression of v-Src in WM793 cells rescued invadopodium formation in STIM1 knockdown cells; n = 49, 46, 49, and 48 for control shRNA, STIM1 shRNA, control shRNA + v-Src, and STIM1 shRNA + v-Src, respectively. (G) Ectopic expression of v-Src rescued the inhibition WM793 cell invasion by STIM1 knockdown. The relative levels of p-Src, p-Akt, and p-FAK in A–D were determined through densitometry using ImageJ software. Horizontal bars in E–G represent means ± SEM. Two-tailed p-values are determined by Mann–Whitney test. The numbers of cells used for quantitation were indicated in the parenthesis of respective figure labeling, and representative results from at least three similar independent experiments were presented. sh, shRNA.