2 7 dichlorodihydro fluorescein diacetate dchf da

The immunofluorescence staining of mouse oocytes was performed as previously described [20 (link)]. Briefly, the spindle and chromosome were stained using anti-mouse β-tubulin antibody (CST, Boston, MA, USA) and Hoechst 33258 (Servicebio, Wuhan, China), respectively. DNA damage levels were evaluated using anti-rabbit phospho-histone H2AX antibody (CST, Boston, MA, USA). For evaluation of intra-oocyte ROS and MMP, the oocytes were stained with the 2′,7′- dichlorodihydro- fluorescein diacetate (DCHF-DA) (Sigma, St. Louis, MO, USA) and MMP detection (JC-1) kit (Beyotime Biotechnology Research Institute, China), respectively. The stained oocytes were observed and photographed under a fluorescence microscope (Carl Zeiss, Batenwerburg, Germany) using fixed microscopic parameters. Image J software (NIH, Bethesda, MD, USA) were applied to measure the fluorescence intensities.

The human cells such as MD-MBA 231, HEK293T, HKC, A549 cells were obtained from the ATCC, DMEM medium, phosphate buffered saline (PBS), Trypsin, and Penicillin/streptomycin were purchased from GIBCO. ATP, MgCl2, Fetal bovine serum (FBS), TBHQ, DMSO were purchased from Sigma. Western Lightning® Plus-ECL kit was purchased from PerkinElmer. Cell Counting Kit-8 was purchased from (Dojindo). Protein Assay kit was purchased from Bio-Rad. QuikChange® site-directed mutagenesis kit was from Stratagene.Nuclear and Cytoplasmic Protein Extraction Kit was purchased Sangon Biotech. The oxidant-sensitive probe 2’,7’-dichlorodihyd rofluorescein diacetate (DCHF-DA) was purchased from Sigma-Aldrich. ARE-luciferase reporter plasmid and pGL6 plasmid were purhcased from Beyotime Institute of Biotechnology. Flag-Keap1, C151S-Keap1, C273S-Keap1, C288S-Keap1, Flag-Nrf2, Gal4-Neh2 and Neh2-luciferase reporter plasmid were kindly gifted from Xiaodong Wang’s Lab (NIBS). The plasmids of HA-ubiquitin, HA-Cul3 were purchased from Addgene. The antibodies of Nrf2 (ab76026), keap1 (ab66620) and Lamin A antibody (ab26300) were purchased from Abcam, the antibodies of HA, Gal4, Flag were purchased from Sigma. The antibodies of HO-1 and NQO1 were purchased from Santa Cruz Biotech.