Fig. 3: NCE-associated Ca²⁺ oscillations induce mitochondrial ATP production.

a Representative images of TMRM fluorescence in HeLa cells stimulated or not with low or high dose EGF after 45 s since recording started. Bar, 10 µM. b Left, time course of TMRM fluorescence intensity in cells treated as in “a”. Right, Total peak area±SD: -EGF/N = 219; LowEGF/N = 217; HighEGF/N = 205; N = number of cells (n = 5). c TMRM fluorescence measured in HeLa cells, subjected to the indicated KD or mock control, and stimulated with high dose EGF (5 min). Mock unstimulated cells (-EGF), negative control. Total peak area is reported. N=number of cells: -EGF/N = 68, Control/N = 421, RTN3-KD/N = 316, RTN4-KD/N = 324, IP3R-KD/N = 265, IP3R-KD/N = 228 (n ≥ 3). d Left, representative TPEF images of HeLa cells mock transfected/unstimulated (Control/-EGF) or subjected to RTN3 KD or mock transfection (Control) and stimulated with high dose EGF (5 min). Images have a dimension of 70 × 70 µm². Scale bar: 10 µm. Right, quantification of the distribution of TPEF signal in HeLa cells treated as in left panel: N, Field of View, -EGF/N = 26, Control/High EGF/N = 32, RTN3-KD/High EGF/N = 27 (n = 3). Statistical significance was calculated using the non-parametric U-Mann Whitney test, two-tailed. e Left, schematic showing luciferase targeted to the PM inner leaflet by fusion with the PM-targeting ___domain of SNAP25. Right, representative IF staining showing the localization of the SNAP25-luciferase fusion protein [SNAP(PM)-Luc] (n = 3). Bar, 10 μm. f Luminescence was measured in HeLa cells expressing PM-Luc stimulated with low or high EGF. Left, representative curves of luminescence over the baseline; cps, count per second. Right, mean AUC ± SD. N=number of coverslips (for panels f–h): HighEGF/N = 11, LowEGF/N = 12 (n = 2). g, h Luminescence was measured in HeLa cells expressing PM-Luc were subjected or not to the indicated KDs (g) or inhibitor treatments (h) and stimulated with high dose EGF. Results are presented as in f. g Control/N = 4, RTN3-KD/N = 4, RTN4-KD/N = 4; a representative experiment of n = 4 is shown. (h) Control/N = 18, MCUi11/N = 12, oligomycin (OMY)/N = 10, xestospongin (XeC)/N = 12 (n ≥ 3). Box plots (panels b–d) are defined as in Fig. 2f. Each Pair Student’s t test, two-tailed (panels b, c, f–h); exact p-values are shown; ns not significant, n biological replicates. Source data are provided as a Source Data file.