Figure 6

Effect of Orai1 knockdown on Ca ²⁺ entry induced by BPA treatments in LNCaP cells ( A, B, C, D). Forty-eight hours prior to recording Ca²⁺ signals by calcium imaging, LNCaP cells were divided into paired groups and transfected with control siRNA (siCTL), or siOrai1 in the presence of 1 or 10 nM BPA. For Ca²⁺ recording, cells were treated with 200 nM thapsigargin (TG) in Ca²⁺-free bath solution (0 Ca²⁺) and exposed to 2 mM extracellular Ca²⁺ (2 Ca²⁺) as indicated. Using the same protocol described above, pharmacological tools were used to study the involvement of Orai1 in BPA-induced SOCE amplification. LNCaP cells were treated with BPA (1nM, 48h) and then the TG-induced SOCE was studied in the presence or absence of BTP2 (2 μM) (E), an inhibitor of Orai1 or SKF96365 (10 μM) (F), a broad spectrum inhibitor of ion channels involved in SOCE. Each experiment was repeated at least 3 times in duplicate in different cell cultures on a field of 25–40 cells and representative experiments performed on 50–80 cells as mean ± S.E. are presented. A quantification of the SOCE for each experiment is presented in (B) and (D) (mean ± S.E., n = 50–80 cells), *P < 0.001. The TG application is shown by an arrow and extracellular Ca²⁺ increases from 0 (0 Ca²⁺) to 2 mM (2 Ca²⁺) are marked by horizontal bars.