Fig. 2: In vitro mechanistic characterization of the mitochondrial effects of harmol. | Nature Communications

Fig. 2: In vitro mechanistic characterization of the mitochondrial effects of harmol.

From: Peripheral modulation of antidepressant targets MAO-B and GABAAR by harmol induces mitohormesis and delays aging in preclinical models

Fig. 2

a, b Western blots of the indicated proteins in differentiated C2C12 myotubes 1 h (a) or 3 h (b) after treatment with 0.1% DMSO, 1.3 μg/ml harmol (H), 30 μM rosiglitazone (R) or 250 μM AICAR (A). Quantifications of the significantly different proteins are shown in the bar graphs to the right (n = 6 independent cell samples for each condition). c Confocal images of differentiated C2C12 cells treated with DMSO (01%), harmol (1.3 µg/ml) or FCCP (500 nM) for 45 min and stained for mitochondria with Mitotracker (Mito); for lysosomes with Lysotracker (Lyso); or for active mitochondria with TMRM. d Quantification of the colocalization of Mitotracker and Lysotracker staining, calculated as the Pearson r correlation coefficient in the same cells and treatments as in (c). e C2C12 myotubes stained with TMRM were treated with the indicated concentrations of harmol alone or combined with 20 µM chloroquine (CQ) for 30 min, and then TMRM intensity was recorded before (T0) and 60 min after (T60) treatment with harmol. f Western blots of the indicated proteins in differentiated C2C12 myotubes 16 h after treatment with 0.1% DMSO, 1.3 μg/ml harmol (H), 30 μM rosiglitazone (R) or 250 μM AICAR (A). (n = 6 independent cell samples for each condition). g Quantitative PCR experiments measuring the ratio between mitochondrial DNA and nuclear DNA (mt/nDNA) in differentiated C2C12 myotubes treated with 1.3 µg/ml harmol or 250 μM AICAR for the indicated times. h ATP content in differentiated C2C12 myotubes treated with 0.1% DMSO or with 1.3 μg/ml harmol was assessed at the indicated timepoints (a.u. arbitrary units). Bars and line-connected dots represent the average of the indicated replicates. Dots in bar graphs (a, b, d, f, g) represent independent cultured cell samples. Each lane in Western blots (a, b, f) represents independent cell samples. Error bars represent the standard deviation. Statistical significance was assessed using the one-way ANOVA (a, b, d, f) or the two-way ANOVA (e, g, h) tests with Tukey’s correction for multiple comparisons. P values are indicated when P < 0.05. Source data are provided as a Source Data file.

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