Kmt5a

Whole cells were washed with ice-cold phosphate-buffered saline, harvested, and resuspended in lysis buffer (Cell Signalling Technology, Danvers, MA). Protein samples were boiled for 10 min at 100 °C in sample loading buffer. Equal amounts of protein (50 μg) from different groups of HUVECs were separated by 8–10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to PVDF membranes (Millipore, Billerica, USA). Membranes were blocked with 5% skimmed milk solution for 1 h, and then all membranes were incubated with primary antibodies at 4 °C for 12 h. The primary antibodies used in the present study were as follows: monoclonal antibodies against β-actin (ProteinTech, Wuhan, China, 60004-1-Ig), KMT5A (ProteinTech, Wuhan, China, 14063-1-AP), CREB (Cell Signalling Technology, Danvers, MA, #9197), PTP1B (ProteinTech, Wuhan, China, 11334-1-AP), p-p65 (Signalway antibody, Maryland, #11014) and H4K20me1 (Abcam, Cambridge, UK, ab9051). After washing the membranes with PBST 5 times, an HRP-conjugated secondary antibody was added for 1 h at room temperature. Then, the membranes were washed five times with PBST, and the protein signal was detected by an ECL system.

Cell protein extracts were obtained using cell lysis buffer (Jiancheng Bio, Nanjing, China). Different groups of HUVECs with equal amounts of proteins (50 μg) were separated by 8–12% SDS-PAGE and transferred to PVDF membranes (Millipore, Billerica, USA). The membranes were incubated in a 5% skimmed milk solution at room temperature for 1 h. Then, all membranes were incubated with the corresponding primary antibodies at 4 °C for 12 h. The primary antibodies used in the present study were as follows: monoclonal antibodies against β-actin (Proteintech, Wuhan, China), KMT5A (Proteintech, Wuhan, China), H4K20me1 (Abcam, Cambridge, UK), ets1 (Proteintech, Wuhan, China), PFN2 (Proteintech, Wuhan, China), αSMA (Proteintech, Wuhan, China), S100A4 (Proteintech, Wuhan, China), CD31 (Proteintech, Wuhan, China) and vimentin (Proteintech, Wuhan, China). Then, an HRP-conjugated secondary antibody was employed. The protein signal was determined by an ECL system (Shanghai Epizyme Biomedical Technology Co. Ltd., Shanghai, China).

The tissues of human subjects and rats were wrapped in paraffin and then processed for HE, Masson staining and IHC. HE was used to detect histopathological abnormalities. Masson staining was used to determine the severity of fibrosis according to the kit instructions (Solarbrio, Beijing, China). The paraffin sections were incubated with antibodies against KMT5A (Proteintech, Wuhan, China), ets1 (Proteintech, Wuhan, China), PFN2 (Proteintech, Wuhan, China), vimentin (Proteintech, Wuhan, China), S100A4 (Proteintech, Wuhan, China), αSMA (Proteintech, Wuhan, China), and CD31 (Proteintech, Wuhan, China) at 4 °C for 12 h.