Mitosox mitochondrial superoxide indicator

Adv-vectors (KLF4, PDGFRA and NAMPT) were used to overexpress target genes, and sh-RNAs (KLF4, PDGFRA and NAMPT) were used to knock down target genes. All were purchased from Viogene Bio. (Jinan, Shandong, China). The KLF4 luciferase reporter plasmid (pGL4-KLF4) was purchased from Yeasen Bio. (Shanghai, China). A fluorescent dual luciferase reporting system and murine leukemia virus reverse transcriptase were obtained from Promega Co. (Mannheim, Germany). The SA-β-gal-kit was obtained from Beyotime (Haimen, China). The MitoSOX Mitochondrial Superoxide Indicator was obtained from Yeasen (Shanghai, China). The anti-p21 antibody was obtained from HUABIO (Hangzhou, China). The anti-PDGF-BB, anti-PDGFRA, anti-PDGFRB, anti-KLF4, anti-NAMPT, anti-PAI-2, anti-uPA and anti-β-actin antibodies and secondary antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). Other reagents were purchased from Sigma (St. Louis, MO, USA).

MitoSOX Mitochondrial Superoxide Indicator (40778ES50, YEASEN) was used to assess mitochondrial ROS. NRVMs were seeded on cover slides of 12-well plates and transfected with ShR-ATP6AP2 or shR-Scr virus vector for 24 h at MOI = 50 on the 4th day. The medium was changed to a complete culture medium and continued for 48 h of PE stimulation or DMSO control. Then added 37 °C pretreated MitoSOX (500 nM) and incubated for 20 min. After PBS cleaning, the nuclei were stained with DAPI. Imaging with fluorescence microscopy (Ex = 579NM, Em = 599 nm).