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. 2014 Apr 17;5(4):e1188. doi: 10.1038/cddis.2014.129

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Copyright © 2014 Macmillan Publishers Limited

This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/3.0/

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InsP3R mediates IRE1α-KD-induced [Ca²⁺]_i alterations and increase ER Ca²⁺ release, leading to cell death. (a) Treatment with InsP3R blocker (2-APB), not RyRs blocker (Dant; dantrolene), blocked the increase of [Ca²⁺]_i in the IRE1α-KD cells. Changes in [Ca²⁺]_i were determined by the Fluo-4 assay. After siRNA transfection for 48 h, 5 μM of Fluo-4 AM in DMEM was added at 37 °C for 60 min. After washing, dantrolene (20 μM), 2-APB (10 μM), and BAPTA-AM (5 μM) were added for 6 h, and 0.5 μM of thapsigargin was treated for 30 min, and then fluorescent signals were captured using a fluorescence microscope and analyzed in ImageJ (N=3 experiments). Data are shown as the mean percentage±S.E.M. IK, IRE1α-KD cells; Tg, thapsigargin (positive control for Fluo-4 assay); BAPTA, BAPTA-AM (negative control for Fluo-4 assay). **P<0.01 and ***P<0.001 versus vehicle (DMSO)-treated control siRNA-transfected cells; ^###P<0.001 versus vehicle (DMSO)-treated IRE1α-KD cells. NS indicates no significant difference. Scale bar=40 μm. (b) RyRs and InsP3R expression in control and IRE1α siRNA-transfected cells was determined by western blotting, with β-actin as a loading control. (c) The viability of the IRE1α-KD cells treated with dantrolene (20 μM), 2-APB (10 μM), and BAPTA-AM (5 μM) was determined by calcein-AM assay. Data shown are the mean percentage±S.E.M. **P<0.01 versus vehicle-treated control siRNA-transfected cells; ^#P<0.05 versus vehicle-treated IRE1α-KD cells. Data were obtained from at least five replicates per group (N=5 experiments). (d) IRE1α-KD cells showed activation of caspase-3 and -9, which was inhibited by 2-APB treatment. fl, full-length form; clev, cleaved (activated) form. β-Actin is a loading control. (e) Xestospongin C, one of InsP3R-specific antagonists, reversed increased [Ca²⁺]_i in IRE1α-KD cells. In all, 2 μM of xestospongin C was treated with Fluo-4 loaded cells for 6 h, and changes in [Ca²⁺]_i were determined by the Fluo-4 assay. XeC, xestospongin C. Representative images are shown. Scale bar=40 μm. (f) Cell viability was analyzed by calcein-AM assay. 2-APB (10 μM) or xestospongin C (2 μM) were treated with IRE1α-KD cells, and then calcein-AM assay was performed. Data are shown as the mean percentage±S.E.M. *P<0.05 versus vehicle-treated control siRNA-transfected cells; ^#P<0.05 versus vehicle-treated IRE1α-KD cells. Data were obtained from at least five replicates per group (N=5 experiments)