Fig. 3

Luteolin boosts mitochondrial respiration and ATP production without affecting mitochondrial mass or structure. a–d Cortical neurons were treated with luteolin (Lut) for 16 h at the indicated concentrations. OCR was quantified using the Seahorse analyzer. Basal respiration, maximal respiration, and oligomycin-sensitive respiration coupled to ATP synthesis were calculated after the sequential injection of oligomycin (1 μM), FCCP (1 μM), and Ant A (0.5 μM) plus Rot (0.5 μM) (n = 6–7 run in triplicate; each point in the graph represents one independent experiment). e Cortical neurons were treated with luteolin (2.5 μM, 16 h), and total ATP levels were quantified by luminescence (n = 6 run in quadruplicate; each point in the graph represents one independent experiment). f, g Neurons were treated with 2.5 μM luteolin for 16 h. Mitochondrial H₂O₂ levels were quantified using MitoPY1 (10 μM, 25 min) in an epifluorescence cell observer microscope. After 10 min of basal reading, mitochondria were challenged with Ant A (2 μM). Representative images show MitoPY1 fluorescence before and after (insets) Ant A addition (scale bar = 25 μm; 10 μm for insert). Two-way ANOVA revealed an effect of Ant A F(1,137) = 65.85, p < 0.0001 (n = 37 −Lut −AntA, n = 38 +Lut −AntA, n = 33 –Lut +AntA, n = 33 +Lut +AntA, from 3 independent experiments). h Representative images of TEM in primary cortical neurons. Insets show an amplified image of mitochondrial cristae that is highlighted in green (scale bar = 500 nm; 150 nm for insert). i–k Mitochondria profile area (in nm²), aspect ratio, and cristae number per mitochondrial perimeter were quantified by TEM (in i and j, n = 23 Ctr, n = 22 Lut 2.5 μM from 3 independent experiments; in k, n = 19 from 3 independent experiments). Each n represents one cell. Statistical significance: ^*p < 0.05, ^**p < 0.01 using non-parametric Kruskal-Wallis or Mann-Whitney test (in b–e), ^****p < 0.0001 using 2-way ANOVA followed by Tukey’s multiple comparisons test (in g); ns = non-significant