STIM1 and Orai1 are crucial for invadopodium formation and activity. (A and B) Scattered dot plot showing the effects of Ca²⁺ chelators and Ca²⁺ blockers on invadopodium number per cell (A) and gelatin degradation area per cell (B). Horizontal bars represent means ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001 as calculated by two-tailed Mann–Whitney test. NS indicates not statistically significant. (C) Representative fluorescence micrographs showing effects of STIM1 and Orai1 shRNA on invadopodium formation and focalized proteolysis in WM793 cells. (D) Scattered dot plot showing that depletion of STIM1 and Orai1 with shRNAs decreased the mean number of invadopodia per cell when compared with WM793 cells expressing control shRNA. (E) Orthogonal views of confocal z stacks showing Orai1 shRNA inhibited gelatin degradation and penetration into Alexa Fluor 488–gelatin coating by invadopodia (arrowheads) in WM793 cells, with WM793 cells expressing control shRNA as a control. Bars: (horizontal) 10 µm; (vertical) 2 µm. (F and G) Scattered dot plot showing that inhibition of SOCE with STIM1 shRNA and Orai1 shRNA inhibited the focalized proteolysis activity of individual invadopodium when compared with control shRNA, as determined by the quantification of IDX. (H) Representative fluorescence micrographs showing that ectopic STIM1 overexpression promoted invadopodium formation and focalized proteolysis of Alexa Fluor 488–gelatin. (I) Scattered dot plot showing that ectopic STIM1 increased numbers of invadopodia per cell, which was abrogated by 100 µM 2-APB; n = 51, 47, 52, and 52 for control, STIM1, control + 2-APB, and STIM1 + 2-APB, respectively. (J) Effects of STIM1 overexpression on the degradation activity of individual invadopodium in WM793 cells. Insets in C and H are magnified views of the boxed areas in the main images. RU, relative unit. Bars (main images) 10 µm; (insets) 2 µm. Two-tailed p-values were determined by Mann-Whitney test or by unpaired Student’s t test after log transformation. Horizontal bars represent means ± SEM. The numbers of cells used for quantitation are indicated in the parenthesis of respective figure labeling, and representative results from at least three similar independent experiments are presented. Ctrl sh, control shRNA.