Fig. 2: Performance of NEMOer sensors in non-excitable mammalian cells.

A Typical ER-Ca²⁺ responses in HEK293 cells induced by ionomycin (iono, 2.5 μM). B, C G-CEPIA1er or NEMOer responses to ER-Ca²⁺-lowering by submaximal receptor stimulation with carbachol (CCh, 10 μM) in HEK293 cells. Initial ER Ca²⁺ decreasing recorded in nominally Ca²⁺ free solution. Left, typical traces; right, statistics. B (**** P < 1E−15, one-way ANOVA). C Typical cells showing ER Ca²⁺ oscillations in 2 mM Ca²⁺ imaging solution. D Simulated F/F₀ vs. [Ca²⁺]_ER, assuming a 20% [Ca²⁺]_ER drop due to ER Ca²⁺ release. The simulation was performed using the Hill equation and in situ parameters from Fig. 1E, with F_min set to 1. E Simultaneous monitoring of 10 μM CCh-induced Ca²⁺ oscillations in ER and cytosol in HEK293 cells. NEMOer-f or G-CEPIA1er was transiently co-expressed with the cytosolic red indicator, R-GECO1.2. Top left, representative response traces; top right, statistics. (***P = 0.0002, unpaired Student’s t-test, two-tailed.) Bottom, averaged response traces of R-GECO1.2 together with corresponding G-CEPIA1er or NEMOer-f (left two panels), and a direct comparison of the responses between NEMOer-f and G-CEPIA1er (right panel). F–H Ratiometric signals of mTurquoise2 - NEMOer-m (TuNer-m) in COS-7 cells collected with multimodality structured illumination microscopy (Multi-SIM). The TuNer ratio (R) is defined as R = F_NEMOer/F_sfTq2ox. (F) Typical basal fluorescence and ratiometric images, with enlarged views. Purple arrows show higher fluorescence with a lower ratio, and yellow arrows indicate the opposite. TuNer-m ratios within ER sheets and tubules. (G) Diagram of Region of Interest (ROI) selection in ratio image: ER sheets (white circles), ER sheets edge (yellow circles) or ER tubules (green circles) (H) Statistics of (F), mean size: sheets 0.06 ± 0.01 μm², edge 0.07 ± 0.01 μm², tubules 0.07 ± 0.01 μm². (****P = 1E−7, **P = 0.0019, ns P = 0.0723, one-way ANOVA, n = 70 ROIs from at least 15 cells). 3 independent biological replicates, ≥ 5 cells per repeat. A–C 3 independent biological replicates, ≥ 15 cells per repeat. (A, B, C, E) Error bar denotes SEM.