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. 2022 Mar 16;9:844671. doi: 10.3389/fcvm.2022.844671

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Copyright © 2022 Wang, Xu, Wu, Shen, Lin, Fang, Zhang, Shen, Liu and Wu.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Role of Orai1-mediated store-operated Ca²⁺ entry (SOCE) in parathyroid hormone (PTH)-induced NFAT nuclear translocation in human umbilical vein endothelial cells (HUVECs). (A–F) Representative confocal microscopy images and summary data (I) showing NFATC1 distribution in HUVECs treated for 24 h with (A) vehicle control, (B) 100 pM PTH, (C) PTH + CsA (calcineurin inhibitor), (D) PTH + W7 (calmodulin antagonist), (E) PTH + BTP2 (an Orai nonspecific inhibitor) or (F) PTH + Orai1 siRNA transfection. Green fluorescence indicates NFATC1; Blue, 4′,6-diamidino-2-phenylindole (DAPI) indicates nuclei. Data are shown as the mean ± SEM; n = 4. **P < 0.01, ***P < 0.001 vs. Con or PTH analyzed by one-way analysis of variance followed by Dunnett's multiple comparisons test. (G,H) Representative Western blot images showing fractionation assay results indicating the presence of p-NFATC1 in the cytoplasmic [Cyto, (H)] and NFATC1 in the nuclear [Nuc, (G)] extracts under the same treatment conditions as for confocal microscopy analyses. Lamin B1 is a nuclear marker; β-Tubulin is a cytoplasmic marker. (I) Summary data showing the ratio of green fluorescence intensity of NFATc-GFP in the nuclear (Nuc)/cytoplasmic (Cyto).