Tunel kit

Mouse kidney tissue was fixed in 4% paraformaldehyde for more than 24 h. The tissue was dehydrated by gradient ethanol and was transparent by xylene. Subsequently, the tissue was embedded in paraffin. A paraffin microtome (YD-315, Yidi, China) was used to prepare 4-μm-thick sections. The slices were baked in a 62°C oven for more than 8 h. The tissue was deparaffinized and rehydrated with xylene and gradient ethanol. The instructions of TdT-mediated dUTP nick end labeling (TUNEL) kit (40306ES50, Yeasen, China) were strictly carried out. The apoptosis of kidney tissues in each group was observed under a fluorescence microscope (BA410T; Motic, Singapore). Here, 3–5 400× visual fields for each group were randomly selected. Apoptosis rate (number of positive nuclei under the field of view/total number of nuclei under the field of view) was evaluated in each group.

The isolated liver tissues were fixed with 4% paraformaldehyde for 24 h. Subsequently, tissues were dehydrated with ethanol and embedded in paraffin. Paraffin-embedded tissues were serially cut into sections with a thickness of 4 μm and used for the following histopathological examination. Hematoxylin–eosin (H&E) staining was conducted to observe the histological changes. Van Gieson (VG) staining was performed to observe the collagen deposition. The sections were stained with hematoxylin and eosin (Aspen, Canada, USA) or VG solution according to the manufacturer's protocols. The histological morphology was observed under a light microscope (Olympus, Japan). Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay was conducted to detect the cell apoptosis rate of liver tissues under the manufacturer's instructions. The cell nucleus was double-dyed using TUNEL kit and DAPI (Yeasen Biotech CO., Ltd., Shanghai, China). The TUNEL-positive cells were visualized using a fluorescence light microscope (Olympus, Japan).

Prior to the assay, CHON-001 cells were treated by 10 ng/ml IL-1β for 72 h. Following PBS washing in the harvested CHON-001 cells, 15 min of immobilization with 4% paraformaldehyde (Shanghai Yeasen Biotechnology Co., Ltd.) was performed at room temperature. Subsequently, prior to DAPI staining, 50 μl TUNEL kit (Shanghai Yeasen Biotechnology Co., Ltd.) was added for 1 h of incubation at 37 °C. Images were acquired under a fluorescence microscope (Olympus).

DMSO, DAPI, and MTT were purchased from Sigma-Aldrich (St. Louis, MO, USA). Propidium iodide (PI)/RNase staining kit and Annexin V-APC/7-AAD kit were purchased from Becton Dickinson (San Diego, CA, USA). The TUNEL kit was purchased from Yeasen Biotech (Shanghai, China). 3-MA was purchased from Selleckchem (Houston, TX, USA). ATRA, AM80, AM580, and THZ-1 were purchased from MedChemExpress (Monmouth Junction, NJ, USA). Antibody information is detailed in Supplemental Methods.

The terminal deoxynucleotidyl transferase-mediated 2′-deoxyuridine 5′-triphosphate nick-end labeling (TUNEL) assay staining was performed following the manufacturer’s instructions. The liver sections were prepared and stained with reagents of a TUNEL kit (Yeasen Biotechnology, Shanghai, China). TUNEL positive cells portion was analyzed.