5 ethynyl 2 deoxyuridine edu staining

In order to study the role of DNMT1 on HPASMCs proliferation, HPASMCs were treated with 2 μM 5-Aza or DNMT1 siRNA for 24 h followed by incubation with 2% CSE for 48 h. Cell proliferation/viability was then assessed with a Cell Counting Kit-8 (CCK8; Promotor, China) and cell cycle detection kit (KeyGEN BioTECH, China) according to the manufacturer’s instructions. CCK8 and cell cycle analyses were completed using an automated spectrophotometric plate reader (PerkinElmer, USA) and flow cytometer (BD Bioscience, USA), respectively. In addition, the proliferation of HPASMCs infected with Ad.NC or Ad.RASEF for 24 h then 2% CSE for another 48 h was measured by cell cycle analysis and 5-ethynyl-2′-deoxyuridine (EdU) staining (RiboBio, China). EdU was labeled with Apollo 567, and cells were observed via fluorescence microscope.

To evaluate the role of MBD2 in the proliferation of HPASMCs, HPASMCs were treated for 24h with MBD2si and then incubated for 24h with 2% CSE. A Cell Counting Kit-8 (Dojindo, Japan) and a cell cycle detection kit (KeyGEN BioTECH, China) were used to assess cell proliferation in accordance with instructions of the manufacturer. An ELx800 Universal Microplate Reader (Bio-Tek Instruments, Inc., Winooski, VT) and a flow cytometer (BD Bioscience, USA) were used to conduct CCK8 and cell cycle analyses, respectively. Furthermore, HPASMCs proliferation was also measured by 5-ethynyl-2′-deoxyuridine (EdU) staining (RiboBio, China). Apollo 567 was used to label EdU, and the cells were examined through a fluorescence microscope.