Confocal microscope imaging system

After treatments, the cells were treated with dil-oxLDL (10 ng/ml, Yiyuan, China) for 30 min. Next, the cells were washed twice by ice-cold PBS and observed using the Zeiss Confocal Microscope Imaging System (Carl Zeiss, Germany).

Paraffin-embedded liver Sects. (4 µm thick) were used for immunohistochemistry (IHC) experiments. The sections were dewaxed, rehydrated, and quenched with 3% H₂O₂, followed by heat-induced epitope retrieval in 10 mM citrate buffer (pH 6) at 95 °C for 20 min. Nonspecific antigens were blocked with 1% BSA (cat: A7906, Sigma-Aldrich). Anti-Ki67 (1:500, ab15580, Abcam) and anti- Hnf4α antibodies (1:500, 3113S, CST) were incubated overnight at 4 °C. Goat-anti-rabbit fluorescein isothiocyanate-labeled IgG or goat-anti-mouse rhodamine IgG (1:200, Proteintech, Rosemont, IL, USA) were incubated at 4 °C for 2 h, followed by 4′,6-diamidino-2-phenylindole (DAPI) staining (Abcam) the cell nucleus. Slides were mounted and visualized using an OLYMPUS microscope.
Primary hepatocytes or macrophages were seeded in a cell culture dish for immunofluorescence experiments. The cells were fixed and permeabilized at 4 °C for 30 min. After incubation with anti-Tet2 (1:100, ab124297, Abcam), anti-Ki67 (1:100, ab15580, Abcam), and anti-Stat1 antibodies (1:100, 9176, CST) at 4 °C overnight, the cells were washed with PBS and stained with goat-anti-rabbit FITC-labeled IgG or goat-anti-mouse rhodamine IgG (1:200, Proteintech) at 4 °C for 2 h, followed by DAPI staining (Abcam). The cells were viewed using a Zeiss Confocal Microscope Imaging System (Carl Zeiss, Jena).

The heart tissue sections were blocked with goat serum at room temperature for 30 min, and then stained with fluorescein conjugated wheat germ agglutinin staining (Alexa Fluor‐488, Invitrogen, CA) was used to evaluate cardiomyocyte size. Myocyte nucleus was stained using 4′, 6‐diamidino‐2‐phenylindole (DAPI, Sigma, USA). For each group, approximately 50–100 randomly chosen cardiomyocytes were analyzed by using Image J software to measure the cross-sectional cardiomyocyte area. To evaluate the expressions of YTHDF1/2/3 proteins in heart tissue, heart tissue sections were blocked, incubated with primary antibodies against α-actinin (Sigma, USA) and YTHDF1/2/3 (Abcam, USA) at 4 °C overnight, and treated with fluorescence-conjugated secondary antibody for 1 h at 37 °C. The nuclei were counterstained with DAPI. After washing, the sections were imaged by a Zeiss Confocal Microscope Imaging System (Carl Zeiss, Germany).