SOCE-mediated Ca²⁺ oscillations regulate invadopodial precursor assembly. (A) WM793 cells were starved overnight, and invadopodial (Invado.) precursor assembly were initiated by stimulation with 10% FBS. Data are presented as means ± SEM (n = 33). (B, C, and E) Representative traces from single cell Ca²⁺ imaging experiments. WM793 cells were stimulated with 10% FBS in the presence of 2 mM Ca²⁺ (B, n = 307), 0 mM Ca²⁺ (C, n = 178), or 2 mM Ca²⁺ and 20 µM SKF96365 (E, n = 364). (D) Representative traces showing restoring the extracellular Ca²⁺ from 0 to 2 mM also restored Ca²⁺ oscillations (n = 150). (F) Scattered dot plot showing quantification of Ca²⁺ oscillation frequencies in control (2 mM Ca²⁺; B), 0 mM Ca²⁺ (C), or 20 µM SKF96365 (E) groups. Each data point represents oscillation frequency in a single cell (means ± SEM; n = 86). ***, P < 0.001 as determined by two-tailed Mann–Whitney test. (G) Live cell imaging to show serum-stimulated assembly of invadopodial precursors in control condition or in conditions that suppressed Ca²⁺ oscillation (0 mM Ca²⁺ and 20 µM SKF96365). Bar, 5 µm. (H) Cells were serum starved overnight, stimulated with 10% FBS under different conditions as indicated, fixed after 30 or 60 min, and stained with phalloidin for invadopodial precursors. *, P < 0.05 as determined by two-tailed Mann–Whitney test. Data presented are means ± SEM. (I and J) Representative trace showing Ca²⁺ oscillations in WM793 cells expressing control shRNA (I, n = 434) or STIM1 and Orai1 shRNA (J, n = 305). (K) Quantification of Ca²⁺ oscillation frequencies in control shRNA (I) or STIM1- and Orai1 shRNA–expressing (J) WM793 cells. Data presented are means ± SEM. (L) Effects of STIM1 and Orai1 shRNA on invadopodium precursor assembly (n = 86). Data presented are mean ± SEM. Cells were treated as described in H. Arrows in B–E, I, and J indicate stimulation with 10% FBS. Ctrl sh, control shRNA. F/F0 is defined as ratio between fluorescence at a given time (F) and fluorescence at time 0 (F0).