Bx53 dp80

After the autopsy, the small intestines (duodenum, jejunum, and ileum) were fixed in 10% formalin and embedded in paraffin according to standard procedures. Formalin-fixed paraffin-embedded tissue blocks were sectioned into 5–8 um thicknesses using a standard rotary microtome (HM-340E; Thermo Fisher, Waltham, MA, USA) and placed on slides. The tissues were deparaffinized using xylene and treated in ethanol (100%, 90%, 70%, and 50%) for 5 min each. The tissue sections were then stained with hematoxylin and eosin (H&E) for histopathology. For immunochemistry, antigen retrieval was conducted using citrate buffer (pH 6.0) at 95 °C for 30 min and at room temperature for 20 min. Subsequently, the sections were incubated with anti-PEDV monoclonal antibody (Median diagnostic, Chuncheon-si, Republic of Korea) overnight at 4 °C. The samples were then labeled with horseradish peroxidase-conjugated anti-rabbit and anti-mouse immunoglobulin G antibodies (Vector Laboratories, Newark, CA, USA) and developed using the 3,3′-diaminobenzidine (DAB; Vector Laboratories, Newark, CA, USA) according to the manufacturer’s instructions. The samples were counterstained with methyl-green, and all slides were visualized and imaged using a light microscope (BX53, DP80; Olympus, Tokyo, Japan).

An EdU Cell Proliferation Image Kit (Abbkine, Wuhan, China) was used for EdU staining assays following the manufacturer’s instructions. SCC-9 cells and CAL 27 cells were seeded on sterile round coverslips in 96-well plates with 5 000 cells per well. The next day, OSCC cells were treated with 1 mmol/L phenformin for 48 h and were then incubated with EdU solution for 2 h in a cell incubator. After removing the medium, 0.1 mL 4% formaldehyde was added to each well and incubated for 15 min at room temperature. The cells were then washed with BSA washing solution three times and treated with PBS with 0.5% Triton X-100 for 20 min, followed by washing with BSA washing solution and staining with Click-iT reaction mixture in the dark for 30 min. After washing the cells with BSA washing solution, one drop of DAPI dye was added to each well, after which the cells were observed using a fluorescence microscope (Olympus BX53-DP80, Tokyo, Japan).

HaCaT cells were incubated in a 24-well plate, treated according to their group, and fixed with 4% paraformaldehyde for 15 min. Cells were permeabilized with 0.2% Triton X-100 for 10 min and blocked with 10% goat serum (Solarbio, Beijing, China) for 1 h. Then, cells were incubated with antibodies against cleaved caspase-9 (AF5244, 1:200; Affinity Biosciences, Cincinnati, OH, USA) and cleaved caspase-3 (9661S, 1:100; CST, Danvers, MA, USA) overnight at 4 °C, followed by incubation with Alexa Fluor 488-conjugated Goat Anti-Rabbit IgG (H+L) (ab150077, 1:1000; Abcam, Cambridge, MA, USA) for 1 h at room temperature. Nuclei were stained with 10 μg/mL 4′, 6-diamidino-2-phenylindole (Sorlabio, Beijing, China). Cells were washed thrice with PBS, cell-climbing slices were removed, and loaded onto microscope slides, and cell images were observed using an ortho-fluorescence microscope (BX53+DP80; Olympus, Tokyo, Japan).

The age-synchronized late L4 larvae or young transgenic adult C. elegans including CF1139 (DAF16:GFP), CL2166 (GST4:GFP), KN259 (SOD3:GFP), LD1008 (SKN1:GFP), TJ375 (HSP16.2:GFP) were raised on NGM dishes with or without 250 μM TLB for 72 h at 20°C. Thereafter, TJ375 mutants were treated by heat stress at 35°C for 2 h, and the fluorescence of C. elegans were observed under a fluorescence microscopy (Olympus BX53 + DP80, Olympus, Japanese) at wavelength with excitation/emission (360/420 nm) filters after the transgenic mutants were anesthetized using (-) -tetramisole hydrochloride (5 mM). The fluorescence of transgenic C. elegans were quantified using the Image Pro Plus 6.0 software.

EdU staining was performed to assess the proliferation of HaCaT cells under different conditions. After treatment according to their group, cells were incubated with pre-warmed 2× EdU working solution (20 μM) at 37 °C for 2 h, fixed with 4% paraformaldehyde (Solarbio) for 15 min, and permeabilized with 0.3% Triton X-100 (Sigma-Aldrich, Shanghai, China) for 10 min at room temperature. Then, the supernatant was discarded and the cells were washed and incubated with 1× Click reaction solution at room temperature for 30 min in the dark. After removal of the reaction solution, cells were incubated with 1 × Hoechst 33242 at room temperature for 10 min in the dark. Images were taken using an ortho-fluorescence microscope (BX53+DP80; Olympus, Tokyo, Japan); the proliferating cells exhibited red fluorescence, whereas their nuclei exhibited blue fluorescence.

AdV5-CMV-GFP-LC3 and GFP-RFP-LC3 were purchased from WZ Biosciences (Jinan, China) and viral infections were performed with the protocol described previously.^{62 (link)} In total, 2 × 10⁵ CAL 27 cells per well were plated in 6-well plates. The next day, the desired amount of virus (MOI = 100) was added to the growth medium without antibiotics. At 12 h, the cells were washed with PBS two times and maintained in growth medium for 24 h. The old medium was then replaced with serum-free medium or growth medium with or without phenformin according to the grouping. After treatment for 6 h, images were observed using a fluorescence microscope (Olympus BX53-DP80).