Extended Data Fig. 6: NEMO performance in resolving Ca²⁺ signals that span a wide range of magnitude.

(a) Dim blue light stimulation induced GECI responses in HeLa cells co-expressing Opto-CRAC and GCaMP6m or NEMOs. Left, typical traces; right, statistics (NEMOm, 1000 ms vs 300 ms, ^****, p = 4.3E-7; GCaMP6m, 1000 ms vs 300 ms, p = 0.0178). To avoid direct activation with 488-nm light, minimal (1%) excitation input at 488 nm was used. Three 15-cycles of 2% 473-nm stimulation light with different exposure time were sequentially applied to photo-induce more Ca²⁺ influxes. 100 ms for the first one, 300 ms for the second, and 10000 ms for the third cycle of photo-stimulation. (b, c) SOCE indicated by GECIs in HEK293 cells bathed in stepwise external Ca²⁺. (B, GCaMP6m vs NEMOm; C, NCaMP7 vs NEMOs). Left, typical traces; right, statistics. (For B right panel, NEMOm, 10 mM vs 3 mM, ^****, p = 9.28E-24; GCaMP6m, 10 mM vs 3 mM, p = 0.7917. For C right panel, NEMOs, 3 mM vs 1 mM, ^****, p = 6.152E-12; NCaMP7, 3 mM vs 1 mM, p = 0.0681.) (d-f) Statistics of panels A-C showing SNR of GECIs. (For D panel, 100 ms, p = 2E-15; 300 ms, p = 1.03E-22; 1000 ms, p = 5.06E-18. For E panel, 0.1 mM, p = 6.95E-23; 0.3 mM, p = 7.01E-26; 1 mM, p = 3.79E-36, 3 mM, p = 1.56E-36; 10 mM, p = 5.85E-37. For F panel, 0.1 mM, p = 1.13E-13; 0.3 mM, p = 5.13E-16; 0.6 mM, p = 8.85E-20, 1 mM, p = 1.44E-21; 3 mM, p = 3.58E-22). (A-C) Paired Student’s t-test, two-tailed. (D-F) Unpaired Student’s t-test, two-tailed. n = 3 independent biological replicates. All data in this figure were shown as mean ± s.e.m.

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