Figure 6

Functional role of Orai1α and Orai1β in agonist-induced Ca²⁺ mobilization and COX activation in BCSC derived from WT or Orai1 KO MDA-MB-231 cells. (A) BCSC were isolated from WT-MDA-MB-231 (WT) or Orai1-KO MDA-MB-231 cells either transfected with empty vector (O1KO) or with TK-promoter Orai1α (O1KO(O1α),TK-promoter Orai1β (O1KO(O1β) or both, as indicated. Representative Ca²⁺ mobilization in response to 100 μM histamine measured using fluo-4. Cells were superfused with HBS containing 1 mM Ca²⁺ and stimulated with 100 μM histamine (indicated by arrow). Representative traces from three cells/condition were chosen to represent the datasets (from left to right, n = 21, 13, 10, 8 and 12; n values correspond to individual mammospheres (8–10 cells analyzed per mammosphere)). Bar graphs are represented as mean ± SEM and were statistically analyzed using Kruskal–Wallis test with multiple comparisons (Dunn's test). ***p < 0.001 as compared to Ca²⁺ mobilization in BCSC derived from O1KO cells. (B) BCSC isolated from WT-MDA-MB-231 (WT) or Orai1-KO MDA-MB-231 cells either transfected with empty vector (O1KO) or TK-promoter Orai1α (O1KO(α)), Orai1β (O1KO(β)) or both (O1KO(α + β)), as indicated, were stimulated with arachidonic acid (AA; 8 μM) in a medium containing 1 mM Ca²⁺ and COX activity was determined as described in Methods. Bar graphs represent COX activity presented as mean ± SEM and expressed µU/mg. Data were statistically analyzed using Kruskal–Wallis test with multiple comparisons (Dunn´s test). ***p < 0.001 as compared to COX activity in non-stimulated cells. ^ΦΦΦp < 0.001 as compared to COX activity in O1KO cells.