Le + WD therapy increased LC3II/LC3I ratio (225 ) and p62 (267 ), and decreased Beclin1 protein levels (54 ), thereby indicating autophagosome accumulation (Fig. 6B). AntiOxCIN4 supplementation in WD-fed mice prevented autophagosome accumulation as measured by the enhance in LC3II/LC3I ratio (by 55 ) along with the maintenance of p62 (112 ) and Becliny1 (92 ) protein levels in comparable levels to the SD groups (Fig. 6B). AntiOxCIN4 per se had no significant impact in autophagic markers in SD-fed mice (Fig. 6B). Mitophagy constitutes an essential cellular method for mitochondrial high-quality control by eliminating dysfunctional mitochondria and sustaining mitochondrial homeostasis [24]. WD-fed mice showed a non-statistically considerable enhance of Parkin (176 ), although no alterations were observed in Pink1 (93 ) protein levels (Fig. 6B). AntiOxCIN4 enhanced Parkin (219 ) and Pink1 (130 ) protein levels in SD-fed mice, but its supplementation in WD-fed mice only induced an upward trend in Parkin levels (by 126 ) (Fig. 6B). Within the Car + WDR. Amorim et al.Redox Biology 55 (2022)(caption on next page)R. Amorim et al.Redox Biology 55 (2022)Fig. 6. Effects of AntiOxCIN4 on auto(mito)phagy markers in WD-fed mice with NAFL phenotype and FFAs-treated human HepG2 cells. (A) Common Western blot result of whole-liver homogenates depicting the protein levels of LC3BI, LC3BII, p62, Beclin1, Parkin, Pink1 and -actin (cytosolic marker) in liver homogenates from WD-fed mice inside the absence/presence of AntiOxCIN4 (two.five mg/day/animal). (B) Protein expression levels of numerous autophagic and/or mitophagic markers from WD-fed mice inside the absence/presence of AntiOxCIN4 (two.five mg/day/animal). These blots had been inverted and contrast-optimized for visualization purposes. Quantification from the bands was performed employing the original blots. Quantification of proteins described above in numerous experiments was normalized to -actin levels. Information are expressed as mean SEM. (C) MS-proteomic analysis of hepatic lysosomal-associated markers and cathepsins-related proteins in WD-fed mice within the absence/presence of AntiOxCIN4 (two.5 mg/day/animal). The blue color represents a lower, whilst the red color represents an increase in protein levels. (D) Cathepsin B activity inside the whole liver homogenate from WD-fed mice in the absence/presence of AntiOxCIN4 (two.five mg/day/animal). (E) mRNA transcript levels of autophagy-related genes (LAMP1, LAMP2, TFEB, ATP6V1a, ATP6V1H, ATP6V0e1) in cells treated with car (BSA) or FFA (24 h, 250 M) in the absence/presence of AntiOxCIN4 (48 h, 100 M).Hemoglobin subunit zeta/HBAZ Protein Accession Data are expressed because the imply SEM (N = 5 per cage for the in vivo study and N = 4 for the HepG2 studies) and the results had been normalized to the control condition (set as = 100 ).Alkaline Phosphatase/ALPL Protein Purity & Documentation Statistically significant compared applying two-way ANOVA followed by Fisher’s LSD test for a number of comparisons (P 0.PMID:24456950 05, P 0.01, P 0.0001 vs SD or Vehicle + BSA); (P 0.05, P 0.01 vs WD or Automobile + FFAs).group, a decreased autophagic flux was correlated using a reduction of the related lysosomal membrane protein two (LAMP2) protein level (Fig. 6C) and cathepsin B activity (78 ) (Fig. 6D) Interestingly, AntiOxCIN4 supplementation prevented a WD-induced reduce in LAMP2 and elevated the levels of M6PR (Fig. 6C), a receptor responsible for the binding and transport of acid hydrolases from Golgi apparatus for the lysosomes. These findings are supported by the larger protein levels of lysosomal hydrolases like CTSS and CTSL.