Figure 1. Evaluation of possible players involved in SOcAMPs in NCM460 cells stably expressing EpacH30.

A- Effect of ionomycin (5μM) in the absence of external Ca²⁺. Addition of 1mM Ca²⁺ resulted in a reduction in the FRET ratio (typical of 5 exp./29 cells). At the end of all experiments forskolin (FSK; 50μM) and IBMX (1mM) were added to obtain a supramaximal FRET response. B- Effect of ionomycin in cells pre-loaded with BAPTA-AM (20μM, 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 1mM Ca²⁺ was significantly reduced as compared to scramble controls (6 exp./33 cells). D- Response to ionomycin in scramble controls (6 exp./97 cells). E-F Summary of experiments in panel 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.