ROS and red-fluorescent labeled active mitochondria assay

The cells were divided into four groups: CON, Si-FKBP5, Si-PPAR-γ, Lv-sh-FKBP5-Si-PPAR-γ, meanwhile, blank control and LPS induction were set up in each group to reflect the regulatory differences of target genes in the pathological environment. After this, we gave cells a 30 min Rosup ROS induction mixture treatment. We uesd a Bain-Marie ROS kit, and the cells were inoculated in 96-well black plates constructed of environmentally friendly materials. Fourteen groups were treated in parallel for each group. After the above treatment was completed, the solution was changed to serum-free MEM medium, and 0.2 μL of DHFH-DA (10 mM) and 0.2 μL of mitochondrial red fluorescent probe working solution (Bain-marie; 50 μL of master mix with 420 μL DMSO were used to make the working solution) were added to each well and incubated for 30 min in the dark. The fluorescence intensities were observed with an automatic enzyme marker after the solution change, where ROS was at 488–525 nm and 579–599 nm were active mitochondria. Another cell crawl was performed at the same concentration, stained, and sealed with DAPI, and morphological images were obtained using a laser confocal microscope.