Fig. 1

Differentiated cells grown in pyruvate show increased mitochondrial function and higher susceptibility to mitochondrial complex III inhibition. a Schematic representation of SH-SY5Y differentiation protocol. SH-SY5Y cells were cultured for five days in high glucose media (25 mM) supplemented with 10% FBS and 10 μM RA, followed by 3 days culture in media devoid of FBS but supplemented with 25 ng/mL BDNF and either with 25 mM or 10 mM glucose or 10 mM pyruvate. b Neurite length was quantified using ImageJ in SH-SY5Y cells differentiated for 8 days (n = 28 ND, n = 32 25 G, n = 30 10 G, n = 34 10 P from 4 independent experiments). c–e SH-SY5Y cells were fixed and imaged by TEM. Number of mitochondria profile per cell and mitochondrial profile area (in μm²) are represented (n = 20 for all conditions from 4 independent experiments). Scale bar = 1 μm. f, g ΔΨ_m was measured using TMRM fluorescent probe in non-quenching conditions (5 nM) in a confocal microscope (f) and total fluorescence was quantified (g) by ImageJ (4 independent experiments). Scale bar: 15 μm. h, i Total levels of ATP were measured by luminescence in differentiated SH-SY5Y or mice primary cortical neurons (as indicated) in the presence or absence of mitochondrial complex III inhibitor Ant A (3.6 μM, 30 min) (4 independent experiments run in triplicates). j–m OCR was quantified using Seahorse flux analyzer and basal, maximal, and oligomycin-sensitive respiration were calculated after the sequential injection of oligomycin (1 μM), FCCP (1 μM), and Ant A (0.5 μM), respectively (5 independent experiments run in quadruplicates). ND = non-differentiated; 25 G = 25 mM glucose; 10 G = 10 mM glucose; 10 P = 10 mM pyruvate. Statistical significance: ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001 using non-parametric Kruskal-Wallis test or two-way ANOVA followed by Sidak’s multiple comparison test (in H and I)