C1086

Apoptosis in the kidney tissues was detected in paraffin sections by in situ TUNEL assays that were performed according to a standard protocol (Beyotime Biotechnology, C1086). Ten to fifteen fields were selected randomly from each tissue section and the number of TUNEL-positive cells were determined per 400× field.

Animal studies were conducted according to guidelines approved by the University of Shihezi Institutional Animal Care and Use Committee. Athymic nude mice were maintained in specific pathogen-free conditions. Animals were randomly distributed into groups. To induce tumor formation, athymic nude mice were subcutaneously infected with 2 × 10⁶ Eca109 cells into the left axillary area. Eca109 cells were steadily infected with pSIH1-H1-copGFP/shR-PLCE1 or pSIH1-H1-copGFP lentivirus after selection of puromycin. After treatment via injection, tumor length and width measurements were obtained with calipers. Tumor volumes were calculated with the following formula: tumor volume (mm³) = (major axial diameter × minor axial diameter 2 × 0.5) thrice weekly. On day 35, tumors were detected by an IVIS imaging system (Caliper Life Sciences, USA), and animals were sacrificed and photographed. Tumors were excised, weighed, photographed, and snap-frozen in liquid nitrogen or formalin-fixed and paraffin-embedded. Paraffin embedded tumor tissues underwent routine histological processing with hematoxylin and eosin (H&E) stain. Cell proliferation in tumors was detected by staining histological sections with a monoclonal antibody against Ki-67 (Zhongshan Biotechnology, ZM-0166); apoptosis was assessed by TUNEL assay (Beyotime, C1086).

Hematoxylin and eosin (H&E) staining and Oil red O staining were performed on paraffin-embedded and frozen liver sections, respectively. The expression of F4/80 (sc-52664, Santa Cruz, USA), TL1A (bs-5092R, Biosynthesis, China), Inducible Nitric Oxide Synthase (iNOS) (18985-1-AP, Proteintech, China), and CD206 (DF4149, Affinity, USA) was observed under confocal microscopy. The terminal deoxynucleotidyl transferase-dUTP nick end labeling (TUNEL) assay (C1086, Beyotime, China) was used to detect hepatocyte apoptosis.

By following the instructions (Beyotime, C1086, China), lung sections were incubated with terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL) reagent containing terminal deoxynucleotidyl transferase and fluorescent isothiocyanate dUTP, followed by antigen retrieval.

The TdT-mediated dUTP nick-end labeling (TUNEL) assay was performed using a commercial TUNEL kit (C1086, Beyotime, China) in the paraffin-embedded brain sections, which was used to assess the neuronal apoptosis according to the instructions of manufacturer. Briefly, after deparaffinized and rehydrated, the brain sections were treated with proteinase K (20 μg/ml, Apoptosis Detection Kit; Merck Millipore, USA) for 30 min at 37°C; then, the brain sections were then incubated with a TUNEL reaction mixture for 60 min at 37°C. All the reactions were performed in the dark. Sections were visualized and captured at 20 × by using a Nikon ECLIPSE 80i (Nikon, Japan).

Paraffin‐embedded brain tissues were sectioned coronally (5 µm thick). Histopathological changes were analyzed by hematoxylin and eosin (H&E) staining (HampE, C0105S; Beyotime), Nissl staining (C0117; Beyotime), and TUNEL staining (C1086; Beyotime) according to the manufacturer's instructions. Pictures were taken using a fluorescence microscope.

The P53 inhibitor pifithrin‐α (S1816, Beyotime) at a concentration of 1 μM⁷² and TPEN (7.5 μM) were added together to mice BNCs for 4 h and detected by the TUNEL assay (C1086, Beyotime). For the TUNEL assay, cells were collected and fixed in 4% paraformaldehyde for 30 min, permeabilized with 0.3% Triton X‐100 for 5 min, and incubated with TUNEL assay solution for 1 h in the dark at 37°C.