STIM proteins are essential for breast cancer cell seeding in a soft extracellular environment. (A) Average intracellular Ca²⁺ responses of SOCE are shown for WT (black, n = 126), S1KO (blue, n = 77), S2KO (green, n = 124) or for two independent clones of S1/S2 dKO (red; trace shows average of the two clones, B6, n = 46; C4, n = 54 cells). (B and C) Quantification (bars show the mean ± SEM) of SOCE (B) and Tg-induced ER Ca²⁺ release (C) for individual cells shown in (A). AUC is the area under the curve from time point 400 to 1,200 s. (D) ER Ca²⁺ was measured by tracking the fluorescence of G-CEPIA following treatment with Tg. Right inset shows a representative trace from WT cell illustrating the Tg-induced change in ER Ca²⁺ (Δ). Left, bars show the mean ± SEM of normalized ΔER Ca²⁺ for WT (n = 57), S1KO (n = 45), S2KO (n = 31), or S1/S2 dKO (B6, n = 42) cells. (E) Normalized alamarBlue absorbance (bars show the mean ± SEM, n = 3) at the indicated days following cell seeding is shown for WT, S1KO, S2KO, or dKO cells. (F) Left, representative images of colonies of the indicated type of cells formed on soft agar with or without embedded fibronectin (FN, orange) after 3 wk. Right, quantification of the average number of individual colonies per field of view, as indicated. Bars show the mean ± SEM from two experiments. The scale bar is 350 or 200 μm, as indicated. (G) Time course of average tumor growth after cancer cell inoculation is shown for the indicated cell types (five or six mice in each group). Note that no tumors were detected in mice injected with S1KO, S2KO, or dKO cells. Statistics: one-way ANOVA with Tukey’s post hoc test; *P < 0.05, **P < 0.01, ***P < 0.001.