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. 2023 Nov 21;223(1):e202305048. doi: 10.1083/jcb.202305048

Figure S3.

Figure S3.

(Related to Fig. 4). Quantification of presynaptic ATP, cytosolic pH, mitochondrial and cytosolic calcium dynamics in control and Sirt3 KD nerve terminals. (A) Average traces of raw luminescence/fluorescence values of control and Sirt3 KD hippocampal nerve terminals expressing Syn-ATP, electrically stimulated with 600 AP at 10 Hz. n = 10–32 neurons. (B) Average traces of cytosolic pH in control and Sirt3 KD terminals expressing cyto-pHluorin, electrically stimulated with 600 AP at 10 Hz. n = 7–8 neurons. (C) Representative vGLUT1-pH traces of a neuron supplied with 5 mM glucose, with or without incubation with 2 µM oligomycin. (D) Fractional retrieval block of vGLUT1-pH. Average retrieval block ± SEM: Glucose, 0.14 ± 0.02, Glucose + Oligomycin 0.40 ± 0.05. n = 8 neurons. (E) Average traces of mito4xi-GCaMP6f showing mitochondrial Ca2+ uptake in control and Sirt3 KD axons stimulated with 20 AP at 20 Hz. (F) Peak responses of Mito4xi - GCaMP6f (ΔF/F) following stimulation. Mean ΔF/F ± SEM: control, 0.34 ± 0.04, Sirt3 KD, 0.26 ± 0.02. n = 19–26 neurons. (G) Average traces of physin-GCaMP6f showing cytosolic Ca2+ uptake in control and Sirt3 KD axons stimulated with 1AP. (H) Peak responses of physin-GCaMP6f (ΔF/F) to stimulation. Mean ΔF/F ± SEM: control, 0.27 ± 0.06, Sirt3 KD, 0.21 ± 0.04. n = 13–20 neurons. (I) Average traces of physin-GCaMP6f showing cytosolic Ca2+ uptake in control and Sirt3 KD axons stimulated with 10 AP at 10 Hz. (J) Peak responses of physin-GCaMP6f (ΔF/F). Mean ΔF/F ± SEM: control, 2.5 ± 0.7, Sirt3 KD, 2.7 ± 0.4. n = 13–20 neurons. Grey bars or arrows denote electrical stimulation. Error bars are SEM. Wilcoxon test (D), Two-tailed, unpaired t test (F, H, and J).