Measurement of Oxidative stress by DHE (dihydroethidium) staining, H2-DCFDA (2′,7′-dichlorodihydrofluorescein diacetate) flow cytometry and Amplex red assay

For DHE staining, Human proximal tubular epithelial cells were seeded in 4-chamber slides. After 48–72 hours of siRNA transfection, cells were briefly washed with PBS and incubated in 1 μM DHE (#D1168 Thermo Fisher) for 30 minutes. Then, cells were quickly washed with PBS for 3 times, fixed in 4% paraformaldehyde for 30 min at room temperature. After fixation, cells were washed again for 3 times and immediately subjected to confocal microscopy imaging. Representative fluorescent images from random regions were taken, and the fluorescence signals of DHE staining area were determined using ImageJ software. Cellular oxidative stress was also measured by H₂-DCFDA dye (#D399, Thermo Fisher). Briefly, the medium was replaced with DMEM containing 1 μM H₂-DCFDA dye for 30 minutes in dark. Then, cells were washed with PBS, collected by 0.25% trypsin solution, fixed in cold 75% ethanol, and stored at −20 °C for 16 hours. Fixed cells were subsequently quantified in a BD Biosciences FACS Calibur (UTHSCSA Flow Cytometry Core) with 10,000 events for total cell population using BD Biosciences Cell Quest software, and the data were analyzed by FlowJo software (Ashland, OR).

To measure the hydrogen peroxide in tissue, we used Amplex Red Hydrogen Peroxide Assay Kit (A12222, Thermo Fisher) and performed the experiments according to the vendor’s protocol. 100 μg of lysates in 100 μl of Kreb’s ringer buffer was used and assay was initiated by adding 100 μl of the same buffer containing 50 μM Amplex red together with 0.1 U/ml horseradish peroxidase. Instant formation of resorufin fluorescence was measured using an EnSpire multimode plate reader at excitation and emission wavelengths 540 nm and 595 nm, respectively. The data were normalized with total protein input and averaged from at least 5 repeats.