Fig. 8: Y040-7904 induces mitophagy through the SIRT1/FoxO3 pathway.

A Immunoblotting studies of the levels of mitophagy-associated proteins following treatment with Y040-7904 at concentrations ranging from 5 to 20 μM for 24 h. B Quantification of mitophagy-associated proteins in (A) was performed using Image J software. Data are presented as mean values ± SD. n  =  4 independent biological replicates. Statistical significance was determined using two-tailed paired ttest. Source data are provided as a Source Data file. C Western blots of the levels of phosphorylated ubiquitin (p-Ser65-ubiquitin) following treatment with Y040-7904 at concentrations ranging from 5 to 20 μM for 24 h. Representative immunoblot from 3 independent experiments with similar results. D Immunoblotting studies of the levels of LC3II/I and PINK1 following treatment with Y040-7904 in si-NC and si-PINK1-transfected HT22 cells. Representative immunoblot from 2–4 independent experiments with similar results. E Immunoblotting studies of the levels of phosphorylated Ser65-ubiquitin (pUb) following treatment with Y040-7904 in si-NC and si-PINK1-transfected HT22 cells. Representative immunoblot from 3 independent experiments with similar results. F Immunoblotting studies of the levels of mitophagy- associated proteins at various time points (6 h, 10 h, 20 h, and 24 h) post Y040-7904 treatment (20 μM). G Quantification of mitophagy-associated proteins after Y040-7904 treatment in F. Data are presented as mean values ± SD. n  =  5–7 independent biological replicates. Statistical significance was determined using two-tailed paired t test. Source data are provided as a Source Data file. H Immunofluorescence images of FoxO3 in control and Y040-7904 (20 μM) treated HT22 cells. Representative fluorescence image from 5 independent captures showing similar data. Scale bar = 10 μm. I Changes in FoxO3 protein in the nucleus and cytoplasm of HT22 cells after Y040-7904 treatment (4 h, 6 h). Representative immunoblot from 3 independent experiments with similar results. J The levels of FoxO3 and LC3 in HT22 cells treated with Y040-7904 or FoxO3 inhibitor AS1842856. K Quantification of indicated proteins in (J). Data are presented as mean values ± SD. n  =  3 independent biological replicates. Statistical significance was determined using One-way ANOVA followed by Dunnett’s multiple comparisons test. Source data are provided as a Source Data file. L The illustration describes the mechanism of Y040–7904-induced mitophagy (created using Adobe Illustrator).