Fig. 1.

Evaluation of possible players involved in SOcAMPs in NCM460 cells stably expressing EpacH30. (A) Effect of ionomycin (5 μM) in the absence of external Ca²⁺. Addition of 1 mM Ca²⁺ resulted in a reduction in the FRET ratio (typical of 5 exp./29 cells). At the end of all experiments forskolin (FSK; 50 μM) and IBMX (1 mM) were added to obtain a supramaximal FRET response. (B) Effect of ionomycin in cells pre-loaded with BAPTA-AM (20 μM, 30 min.; typical of 6 exp./49cells). (C) Knockdown by RNAi of the Orai1 channel did not alter the response to ionomycin, however, the inhibitory action of 1 mM Ca²⁺ was significantly reduced as compared with scramble controls (6 exp./33 cells). (D) Response to ionomycin in scramble controls (6 exp./97 cells). (E, F) Summary of experiments in C and D: (E) Amplitude (Δ) and slope of the ionomycin response expressed as percentage of the response in scramble controls. (F) Response to 1 mM Ca²⁺ expressed as a % of the ionomycin response in scramble and siOrai1-treated cells. (G) Effect of overexpression of Orai1 on the response to ionomycin (5 exp./25 mCherry cells/7 control cells). (H) Summary of Orai1 overexpression experiments. Amplitude and slope of the ionomycin response expressed as a percentage of the response in control cells. (I) Summary of Orai1 + STIM1 overexpression experiments. (5 exp./9 control cells/10 mCherry cells) **P < 0.02, ***P < 0.0001.