Reactive oxygen species ros kit

After being cultivated on 12-well plates for 24 h at a density of 1×105 cells/well, the cells were stimulated with 10, 30, and 60 μg/mL of TE. After 2 h of incubation, the intracellular ROS generation was assessed according to the reactive oxygen species ROS kit (Beyotime, China). The fluorescent probe DCFH-DA was diluted in serum-free medium (DMEM) to a final concentration of 10 μM/L. Cells were collected and suspended in diluted DCFH-DA, incubated at 37°C for 20 minutes with appropriate inversion and mixing, so that the probe and cells were in full contact with each other. The ratio of fluorescence intensity was performed using a CytoFLEX flow cytometer (Beckman Coulter, Brea, CA, USA) with emission and excitation wavelengths of 488 and 525 nm, respectively. In addition, 50µM DHE fluorescent probe staining solution was prepared and mixed with the treated cells, which were detected by fluorescence microscope (Rockford, IL, USA) under excitation wavelength of 510 nm and 600 nm emission wavelength. The imageJ was used to analyze the quantitative of relative fluorescence intensity. For drug intervention, HaCaT cells were treated with mixed solutions containing TRX (1 mM) and TE (10 μg/mL) and incubated for 2 h, similar to the previous detection method to test TRX inhibition on TE-induced oxidant stress.

MiR-93 mimic, inhibitor and matched negative control (NC) were synthesized by GenePharma, Shanghai, China. Lactate dehydrogenase (LDH), creatine kinase (CK) and malondialdehyde (MDA) commercial kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Reactive oxygen species (ROS) kit, Annexin V-FITC/propidium iodide (PI) apoptosis kit, and LY294002 were purchased from Beyotime Institute of Biotechnology (Shanghai, China). PTEN, glucose-regulated protein (GRP) 78, C/EBP homologous protein (CHOP), caspase-12, P-AKT, Bax, Bcl-2, caspase-3 and β-actin antibodies were obtained from Santa Cruz Biotechnology (USA). HiPerFect Transfection reagent and miRNeasy Mini kits were from Qiagen (Germany). Quick-change Mutagenesis Kit were from Stratagene (Germany). A dual luciferase psiCheck-2 reporter plasmid and dual luciferase reporter assay kit were from Promega (USA).