Cyanine3 (cy3)

H9c2 rat cardiomyocyte cell line was get from Wuhan Punosi Life Science and Technology Co., Ltd (Wuhan, China). DMEM low-sugar medium and FBS was purchased from HyClone (Logan, UT). The CCK-8 was purchased from Dojindo (Kumamoto, Japan). LDH, CK-MB, ROS, and GSH test kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Iron assay kit and GPX4 primary antibodies were purchased from Abcam (Cambridge, USA). Rabbit anti-rat primary antibodies DNMT-1 (catalog number 24206–1-AP), GPX4 (catalog number 67763-1-Ig), P62 (catalog number 18420-1-AP), Beclin-1 (catalog number 11306-1-AP), GAPDH (catalog number 60004-1-Ig), and Cy3- (catalog number SA00009-2) and FITC- (catalog number SA00003-2)-labeled goat anti-rabbit secondary antibodies were obtained from ProteinTech (Wuhan, China). Rabbit anti-rat primary antibodies NCOA4 (catalog number A5695) and FTH (catalog number A19544) were obtained from ABclonal (Wuhan, China). Second antibodies were purchased from LI-COR Biosciences (IRDye 800CW; catalog number 926-32219, LI-COR Corporate, USA). DNMT-1 inhibitor 5-aza-CdR were purchased from Selleck (Houston, USA). Lipofectamine 2000 was obtained from Invitrogen (San Diego, USA).

The OEC were rinsed three times in phosphate-buffered saline (PBS) before being fixed in 4% PFA for 20 min, and then the cells were rinsed another three times in PBS. For immunofluorescence, the cells were pre-incubated with 10% goat serum albumin (Sigma) for 60 min at room temperature and rinsed three times in PBS, followed by a 12 h incubation at 4 °C with a rabbit anti-rat P75NTR monoclonal antibody (1:50, Proteintech, Chicago, IL, USA). The secondary goat anti-rabbit antibodies Cy3 (Proteintech) were used at 1:500, and the cells were incubated for 60 min at room temperature. At the 55th min, DAPI (100 ng/mL) was added. Then, the cells were rinsed three times in PBS before being mounted in 50% glycerol. Under 200× magnification, 5 different microscopic fields (200×) were randomly chosen. The number of OEC in each field was calculated and summed to obtain an average number of OEC in each group.

Newly isolated C. sinensis adults were fixed with 4% formaldehyde, embedded in paraffin wax and sliced into 3–5 μm-thick sections using a microtome (Microm, Germany). The sections were then dewaxed and dehydrated. The slides were blocked with goat serum overnight at 4°C and incubated with rat anti-rCsACAT sera (1:400 dilutions) at room temperature for 2 h. Sera from naïve rats (1:400 dilutions) were used as a negative control. The slides were washed with phosphate buffered saline (PBS) containing 0.1% Tween-20 (PBST) three times and then incubated with goat anti-rat IgG labeled with red fluorescent Cyanine dye 3 (1:400 dilutions, Cy3, Proteintech, USA) at room temperature for 1 h in the dark. 0.1% bovine serum albumin (BSA) in PBST was used as dilution buffer. After washing with PBST, fluorescence microscopy (ZEISS, Goettingen, Germany) was used in the visualization of antibody staining.