Foxo1

Hippocampal tissues were lysed using RIPA lysis buffer (Thermo Fisher Scientific, Inc.). Protein concentrations were determined using a BCA assay (OriGene Technologies, Inc.). A total of 25 µg extracted protein was loaded on a 12% SDS-gel, resolved using SDS-PAGE and electrophoretically transferred to a PVDF membrane (Bio-Rad Laboratories, Inc.). The membranes were blocked using 5% non-fat milk for 1.5 h at room temperature. Subsequently, the membranes were washed three times with PBS/0.2% Tween-20 (PBST) for 3 min each time, and incubated overnight at 4°C with rabbit anti-mouse antibodies. The primary antibodies used were: Bcl-2 (cat. no. ab196495; 1:1,000; rabbit polyclonal; Abcam), Bax (cat. no. ab32503; 1:1,000; rabbit monoclonal; Abcam), CC-3 (cat. no. ab49822; 1:1,000; rabbit polyclonal; Abcam), Sirt1 (cat. no. ab189494; 1:1,000; rabbit monoclonal; Abcam), FoxO1 (cat. no. ab52857; 1:1,000; rabbit monoclonal; Abcam) and β-actin (cat. no. ab8227; 1:1,000; rabbit polyclonal; Abcam). After washing three times for 5 min using PBST, the PVDF membranes were incubated with IRDye^® 800CW goat anti-rabbit IgG secondary antibody (cat. no. ab216773; 1:10,000; Abcam) at room temperature for 2 h. Signals were visualized using enhanced chemiluminescent reagent (Bio-Rad Laboratories, Inc.). Densitometry analysis was performed using ImageJ (Image Lab 4.1; National Institutes of Health).

ChIP-qPCR was performed essentially as described by Sinkkonen et al.⁵¹ (link) In brief, TM cells were crosslinked with 1% formaldehyde while gently shaking for 15 min, and the DNA was sheared to fragments ranging from 200 to 400 bp by sonication. The genomic fragments were precipitated using antibodies against ETS1 (1:50; Cell Signaling Technology [CST], Danvers, MA, USA), Foxo1 (1:50; Abcam, Cambridge, MA, USA), and KDM3A (1:50; Abcam). The primers for the amplification of GDF7 were designed according to the four predicted binding sites detected in the reverse ChIP assay (details in Table S7). ChIP-qPCR was run in triplicate, and each ChIP reaction was repeated twice to confirm the reproducibility of results. The results were normalized to the input DNA amplifications.

Total protein was extracted from the rat heart tissues using M-PER mammalian Protein Extraction Reagent (Thermo Scientifics, USA). Protein lysates were boiled for 10 minutes and loaded onto a 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gel. Proteins were transferred to polyvinylidene fluoride (PVDF) membranes and blocked with 5% non-fat milk. Membranes were then incubated overnight at 4 °C with primary antibodies against PPARα (Abcam, UK), FoxO1 (Abcam, UK), phosphorylated FoxO1 (Cst, USA), Akt (Cst, USA), Gck (Abcam, UK), Glut1 (Abcam, UK), Bcl-2 (Abcam, UK), or Bax (Proteintech, China). After washing with Tris buffered saline Tween (TBST), membranes were incubated with secondary antibodies (1:10,000, Proteintech, China) for 1 hour at room temperature (RT), washed again with TBST, and visualized with enhanced chemiluminescence reagents. Chemiluminescence images were obtained by using the ChemiDoc Imaging Systems (Bio-rad) and Image J software was used to quantify protein expression.

The protein isolation of the tm HT-22 cells was performed by applying RIPA lysis buffer (Beyotime) supplemented with protease inhibitor. The protein samples (20 µg) were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to separate it. After the polyvinylidene difluoride (PVDF) transfer, the cells were blocked with 5% skim milk for 2 h at room temperature. The cells were then cultivated overnight with primary antibodies at 4 ℃ and exposed for 2 h to goat anti-Rabbit IgG H&L (HRP) secondary antibodies (cat. no. ab6759; 1:5,000; Abcam) at room temperature. An enhanced chemiluminescence (ECL) kit was used, and Image J Software (NIH, Bethesda, MD, USA) was used to analyze the band intensities. The following primary antibodies were used: PDK1 (cat. no. ab202468; 1:2,000; Abcam), p-FoxO1 (cat. no. ab131339; 1:1,000; Abcam), FoxO1 (cat. no. ab179450; 1:1,000; Abcam), Bcl2 (cat. no. ab32124; 1:1,000; Abcam), Bax (cat. no. ab32503; 1:1,000; Abcam), MMP2 (cat. no. ab92536; 1:1,000; Abcam), vimentin (cat. no. ab92547; 1:1,000; Abcam), E-cadherin (cat. no. ab40772; 1:10,000; Abcam), VEGFA (cat. no. ab46154; 1:1,000; Abcam), and VEGFAR2 (cat. no. #96981; 1,000; Cell Signaling Technology).