Fig. 2.

Orai1 silencing does not significantly reduce the magnitude of the store-operated calcium entry response in PAECs. A: PAECs were engineered for doxycycline-inducible expression of Orai1 shRNA, as described previously (15). Cells were treated with doxycycline (3 μg/ml) every 24 h for a total of 72 h. After doxycycline exposure, cell lysates were collected and subjected to Western blot analysis. Conditional expression of Orai1 shRNA silenced Orai1 protein by 72 h (left), an effect that resulted in an ∼80% decrease in protein as determined by densitometry (middle, n = 20 experiments). Doxycycline treatment induced uniform mCherry expression, consistent with uniform Orai1 shRNA expression (right). B: Orai1 expression was not a determinant of the cytosolic calcium response to thapsigargin in the recalcification protocol following readdition of 100 μM [+Orai1 (n = 4) and −Orai1 (n = 8)], 500 μM [+Orai1 (n = 6) and −Orai1 (n = 10)], and 5 mM [+Orai1 (n = 7) and −Orai1 (n = 9)] calcium, respectively. ns, Not significant. C and D: cytosolic calcium response to 30 nM, 100 nM, and 1 μM thapsigargin in the presence of 2 mM extracellular calcium in the presence and absence of Orai1. C: Orai1 silencing delayed the rise in cytosolic calcium. Pearson P < 0.05 at 30 and 100 nM thapsigargin. *P < 0.05. D: averaged responses following 30 nM [+Orai1 (n = 6) and −Orai1 (n = 7)], 100 nM [+Orai1 (n = 7) and −Orai1 (n = 8)], and 1 μM [+Orai1 (n = 9) and −Orai1 (n = 7)] thapsigargin, respectively.