Fig. 4

Mitochondria-ER contacts are increased by luteolin regulating mitochondrial respiration in neuronal models. a Representative TEM images from primary neurons evidence mitochondria (in yellow) in close contact with ER (in purple) (scale bar = 80 nm) (upper panels). Representative confocal images of differentiated 10 P SH-SY5Y cells transfected with SPLICS_S (in green), and f-actin labeled with phalloidin (gray) (middle and lower panels) (scale bar = 15 μm). b, c Number of MERCS per mitochondria profile and MERCS length were quantified by TEM (n = 31 Ctr, n = 32 Lut 2.5 μM from 4 independent experiments). d Differentiated 10 P SH-SY5Y cells were transfected with SPLICS_S and treated with luteolin for 16 h, and the number of green dots quantified. Each dot indicates a contact between mitochondria and ER (n = 15 Ctr, n = 18 Lut 2.5 μM from 4 independent experiments). e, f Schematic representation of proteins in MERCS (in e). MERCS proteins were quantified by western blotting using specific antibodies. Proteins were extracted from neurons treated for 16 h with DMSO or luteolin (in f) (n = 4–5; each point in the graph represents one independent experiment). OMM: outer mitochondrial membrane; IMM: inner mitochondrial membrane. g–i Cortical neurons (in g, h) or differentiated 10 P SH-SY5Y cells (in i) were treated with DMSO or luteolin (2.5 μM) for 24 h and incubated, when indicated, with XeC (3 μM) for 30 min. OCR was measured using the Seahorse flux analyzer. Spider chart lines represent the fold increase in OCR considering basal respiration of the control cells equal to 1 (n = 6–8 run in quadruplicate; each point in the graph represents one independent experiment). SRC: spare respiratory capacity. Statistical significance: ^*p < 0.05, ^****p < 0.0001 using non-parametric Mann-Whitney test (in b, d); ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 using non-parametric Kruskal-Wallis (in h, i)