Mef2d

H9c2 cells were stimulated with 5% oxygen (O2) and 5% carbon dioxide (CO2) and 90% N2 for 24 h. Next, H9c2 cells were xed with 4% paraformaldehyde for 15 min and incubated with using 0.15% Triton X100 for 15 min at room temperature. H9c2 cells was incubated with MEF2D (1:500, abcam) and HDAC5
(1:500, Cell Signaling Technology, Inc.) at 4˚C overnight after blocking with 5% BSA for 1 h. hASMCs was incubated with goat anti-rabbit IgG-cFL 555 antibody (1:100) for 2 h at room temperature and stained with DAPI for 15 min and washed with PBS for 15 min. The images of hASMCs obtained using a Zeiss Axioplan 2 uorescent microscope (carl Zeiss AG, Oberkochen, Germany).

Aneurysm tissue or cells samples were lysed with ice-cold RIPA buffer with complete protease and phosphatase inhibitors. The protein concentrations were measured using BCA protein assay kit. Total proteins were separated by SDS-PAGE and transferred onto polyvinylidene di uoride (PVDF) membranes. The membranes were incubated with primary antibodies: MEF2D (1:1000, abcam), HDAC5 (1:1000, Cell Signaling Technology, Inc.) and β-Actin (1:5000, Santa Cruz Biotechnology) after blocking with 5% BSA in TBS, followed by incubation with peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology). The signals were detected with the ECL system and exposed by the ChemiDoc XRS system with Image Labsoftware (Bio-rad).