Gsk 3β

Proximity ligation assay was performed with Duolink In Situ Starter Kit (Sigma) according to manufacture’s protocol as described previously^{5 (link)}. The slides were stained with a combination of LSD1 (Cell Signaling Technology), GSK3β (Santa Cruz Biotechnology) antibodies, as well as an IgG control antibody (Sigma) The slides were then examined using a Leica TCS SP2 confocal laser scanning microscope (Leica Microsystems GmbH, Wetzlar, Germany) and the numbers of interaction signal was quantified using the ImageJ software

The isolation of exosomes was approved by western blot analysis using the antibody of CD63 (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Also, the phosphorylation of GSK3β at Ser-9 position was determined in the cells treated with the doses of 2 and 4 µg/mL the pooled exosomes obtained from normal-weight (PN-Exo) females and PO-Exo as well as in the cells treated with the dose of 4 µg/mL obese exosome (O-Exo) and normal-weight exosome (N-Exo) using the western blot analysis. Briefly, the cells were lysed by RIPA buffer (50 mM Tris–HCl, 1% Triton X-100, pH 7.4, 0.2% SDS, 0.2% sodium deoxycholate, 1 mM PMSF and 1 mM Na-EDTA) containing protease inhibitor cocktail and then, the cell lysate was fractionated using SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane. Five percent non-fat dry milk was applied to blocking. Immunoblots were incubated with antibodies against GAPDH, p-GSK3β, and GSK3β (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 18 h, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies for 60 min. The bound proteins were observed by chemiluminescence using enhanced electrochemiluminescence (ECL) reagents and subsequent autoradiography. Eventually, the protein bands were quantitated using ImageJ software.

A 20 mg piece of frozen mixed ventricle was homogenized in 125 μL of sucrose buffer for subsequent extraction of the mitochondrial, nuclear, and cytosolic fractions [39 (link)]. Proteins were assayed as previously described [40 (link)] and incubated for 16 h with primary antibodies (diluted 1:100 to 1:4000) against GCLC, GCLM (provided by Dr. Forman), Nox4, Vdac1 (Abcam, Cambridge, MA, USA), NF-κB p65, acetylated-NF-κB (Lys 310), IκBα, p-IκBα, H3, (Cell Signaling, Danvers, MA, USA), Keap1, Nrf2, GSK3β, Parkin, GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA), 4-hydroxy-2-nonenal (4HNE), Nox2 (EMD Millipore, Burlington, MA, USA), and Hmox1 (Proteintech, Rosemont, IL, USA). Blots were visualized using an Odyssey system (LI-COR Biosciences) and quantified using ImageJ. Nuclear and cytosolic extractions were tested for purity against H3 and GAPDH [39 (link)]. Mitochondrial extractions were also tested for purity with Vdac1 and GAPDH. In addition to consistently loading the same amount of total protein per well, values were further normalized by correcting with the densitometry values of Ponceau S staining [41 (link)]. Three Western blots were run per protein to include all available biological replicates. All blots contained no less than 2 representative samples per group.

N-Nitrosodiethylamine (DEN, Cat:N0258), GoTaq Green Master Mix (Cat:M712B) were purchased from Sigma Aldrich and Promega respectively. TRIzol reagent (Cat:15596026), Verso c-DNA synthesis kit (Cat:AB1453A) were purchased from Thermo Fisher Scientific, Inc.(USA). Primary antibodies β-Catenin (Cat: SC-7963), Wnt3 (Cat: ab50341), PCNA (Cat: SC-56), Gli1 (Cat: SC-20687), Gli2 (Cat: SC-28674), sFRP1 (Cat: ab126613), MMP9 (Cat: SC-393859), GSK3β (Cat: SC-9166), Shh (Cat: SC-9024), CyclinD1(Cat:SC-20044), CyclinB1(Cat:SC-752) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). A horseradish peroxidase (HRP) conjugated goat anti- rabbit IgG, goat anti-mouse IgG secondary antibodies (Cat: ab97200 and ab97265 respectively), Rabbit Specific HRP/DAB (ABC-IHC) Kit (Cat: ab64261) were purchased from Abcam.