Dhe fluorescent probe

Dihydroethidium (DHE) fluorescent probe (Sigma-Aldrich) was utilized to evaluate the renal ROS levels following the previously reported protocol [36 (link)]. Briefly, the frozen sections were stained with DHE (50 uM) for 30 min at 37 °C. After three washing in PBS, the images of renal sections were photographed using a fluorescent microscope (TE-2000, Nikon).

Alda-1, the specific activator of ALDH2, necrostatin-1 (Nec-1), the specific inhibitor of necroptosis and the dihydroethidium (DHE) fluorescent probe were obtained from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). The mitochondrial ALDH2 activity assay kit was obtained from Abcam (Cambridge, UK). The ALDH2 antibody was obtained from Santa Cruz Biotechnology, Inc., (Dallas, TX, USA). The RIP1 and RIP3 antibodies were obtained from Abcam. The mixed lineage kinase domain like pseudokinase (MLKL) antibody was obtained from Affinity Biosciences (Cambridge, UK). The cleaved caspase-3 antibody was obtained from Cell Signaling Technology, Inc., (Danvers, MA, USA). The GAPDH antibody was acquired from Boster Biological Technology Co., Ltd. (Wuhan, China). A Cell Counting Kit-8 (CCK-8) assay kit was from Bestbio Life Technology (Shanghai, China; http://www.bestbio.com.cn; cat. no. BB-4202-1). Primers for ALDH2, RIP1, RIP3, MLKL and GAPDH were acquired from Sangon Biotech Co., Ltd. (Shanghai, China; sequences are presented in Table I). A RevertAid RT Reverse Transcription kit was purchased from Thermo Fisher Scientific, Inc., (Waltham, MA, USA). The SYBR^® Premix DimerEraser™ (Perfect Real Time) was acquired from Takara Bio, Inc. (Otsu, Japan).