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

Figure 3.

Figure 3.

Sirt3 is expressed in neuronal mitochondria and is present in presynaptic terminals. (A) Control hippocampal neurons and neurons transduced with lentivirus expressing Sirt3 shRNA (Sirt3 KD) were immunostained with anti-Sirt3 antibody and the Hoechst nuclear stain. (B) Quantification of Sirt3 immunofluorescence in control and Sirt3 KD neurons. Normalized mean Sirt3 intensity per coverslip ± SEM: Control, 1.02 ± 0.19; Sirt3 KD, 0.34 ± 0.04. n = 4–5 coverslips. (C) Co-immunostaining of neurons with antibodies against Sirt3 and the mitochondrial marker TOMM20. (D) Quantification of Sirt3 colocalization with TOMM20 or Hoechst in neuronal cell bodies. Mean Pearson’s correlation coefficient (r) ± SEM: Sirt3/TOMM20, r = 0.55 ± 0.01, Sirt3/Hoechst: −0.09 ± 0.02. n = 78 cell bodies. (E–G) Coimmunostaining of neurons with antibodies against the neurite marker Tuj1 (E) or presynaptic marker vGLUT1 (F and G). (G) Magnification of the boxed area in F. Arrowheads indicate colocalization of Sirt3 with presynaptic terminals. (H) Quantification of Sirt3 colocalization with vGLUT1 or Hoechst across fields of view (FOVs). Mean Pearson’s correlation coefficient ± SEM: Sirt3/vGLUT1: r = 0.60 ± 0.02, Sirt3/Hoechst: −0.01 ± 0.02. n = 27 FOVs. Scale bars, 10 μm. Mann–Whitney U test (B, D, and H). See Table 1.