Fig. 5.

SOCE mediated by STIM-Orai contributes to the refilling and maintenance of ER Ca²⁺ store, but it is not essential for the long-term homeostasis of ER Ca²⁺ levels. ER Ca²⁺ content was measured with G-CEPIA1er or R-CEPIA1er transiently expressed in HEK WT, STIM KO, or Orai-KO cells. a, b Compared to WT cells, resting ER Ca²⁺ levels in S1KO, SKO, or Orai-KO cells are significantly reduced, while STIM2 KO (S2KO) have no effect on ER Ca²⁺ content. a Typical traces showing the entire time course of recordings used to calibrate ER Ca²⁺ levels in HEK WT, SKO, or Orai-KO cells. b Statistics showing the effect of STIM or Orai-KO on ER Ca²⁺ levels (n > =4, **p < 0.01; ***p < 0.0001; paired t test). c, d As reflected with changes in R-CEPIA1er fluorescence, SOCE process is essential for the acute refilling of ER Ca²⁺ store in HEK cells. In WT cells, after store depletion induced by 100 μM CCh, extracellular addition of Ca²⁺ will result in an increase in Ca²⁺ levels. In HEK SKO or Orai-KO cells that are devoid of SOCE, there is no refilling of ER Ca²⁺ store during the entire period of recording. c Typical traces. Left panel, WT cells; right panel, HEK SKO or Orai-KO cells. d Statistics. (n = 3, p < 0.0001, t test). e, f SOCE process is not essential for the long-term maintenance of ER Ca²⁺ levels. After brief store depletion by 10-min incubation in nominally Ca²⁺-free HBSS solution containing 100 μM carbarcol (CCh) or 2.5 μM ionomycin, cells were then cultured in regular DMEM for another day. Afterwards, the ER Ca²⁺ levels indicated by G-CEPIA1er fluorescence were measured and calibrated. e Representative traces showing the effect of 10-min CCh-induced store depletion on ER Ca²⁺ levels after 24 h regular culture in HEK SKO cells. f Statistic showing 10-min CCh or ionomycin treatments significantly increased the ER Ca²⁺ levels in HEK WT, SKO, or Orai-KO cells 24 h later. (n = 4, p < 0.0001; paired t test). Note: CCh or ionomcyin were then washed away after a 10-min exposure for treatments in e and f. All the data are presented as mean ± SEM