Alexa fluor 488

The sections of the aortic root were prepared and incubated with rabbit anti-HDAC11 (1:100, Abcam, ab18973) and mouse anti-CD31 (1:100, Proteintech, Wuhan, China, 66065-2-Ig), overnight at 4 °C. The HUVECs were fixed with 4% paraformaldehyde solution for 30 min, permeabilized with 0.3% Triton X-100 for 30 min, and then blocked with normal goat serum for 1 h at room temperature. Subsequently, the samples were incubated with anti-HDAC11 antibody (1:100, Abcam, ab18973) or anti-ERG antibody (1:50, Proteintech, 14356-1-AP) overnight at 4 °C, followed by incubation with goat anti-rabbit IgG (H&L) - Alexa Fluor 594 (1:200, ImmunoWay Biotechnology Company, Plano, Texas, USA, RS3611) and goat anti-mouse IgG (H&L) - Alexa Fluor 488 (1:200, ImmunoWay Biotechnology Company, RS3208) for 2 h at room temperature. Nuclei were stained with DAPI (Beyotime, Haimen, China). The samples were observed under a fluorescence microscope.

Tetra were from Sigma-Aldrich, Co. (T1512, St. Louis, MO, USA). Pifithrin-α hydrobromide was from MedChemExpress (HY-15484, Monmouth Junction, NJ, USA). Recombinant Human TGF-β1 Protein was from R&D Systems, Inc. (Minneapolis, MN, USA). The following primary antibodies were used: rabbit anti‐TNAP (ab108337, Abcam, Cambridge, UK), rabbit anti‐α-SMA (ab124964, Abcam), rabbit anti‐Vimentin (ab92547, Abcam), rabbit anti‐Fibronectin (ab2413, Abcam), rabbit anti‐TGF-β1 (SAB4502954, Sigma-Aldrich, Co.), rabbit anti-p-Smad2 (Ser465/Ser467) (#18338, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-Smad2 (#5339, Cell Signaling Technology), rabbit anti-Smad3 (#9523, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-AMPKα1/2 (phosphoThr183/172, YT0216, ImmunoWay Biotechnology, Co., Plano, TX, USA), rabbit anti-p-AMPKα1/2 (YP0575, ImmunoWay), rabbit anti-p53 (ab26, Abcam), rabbit anti-cyclinE (YT1176, ImmunoWay), and mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (YM1038, ImmunoWay). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit (RS0004) or anti-mouse (RS0001) were from ImmunoWay. Goat anti-rabbit IgG secondary antibodies Alexa Fluor® 488 (RS23220, ImmunoWay) were used for immunofluorescent analysis.