Bz x700 fluorescence microscope

Cells (3 × 10⁵ cells/mL) were plated in duplicate in 6-well plates, grown to 70%–80% confluence, and incubated with high-glucose DMEM that contained AG1478 (0 [control] or 0.5 μM) and AuNPs (0 [control] or 1 nM). After 24 h, cells were exposed to irradiation (0 [control] or 4 Gy). After 48 h, cell monolayers were scraped with sterile 200 μL disposable plastic pipette tips and washed with PBS. Wound healing was checked with microscopy at 0, 4, 8, 12, 16, 20, and 24 h at 37°C after wounding. Images were taken with a BZ-X700 fluorescence microscope (Keyence Corporation; 4x magnification), and area was calculated by an analysis software BZ-H4M (Keyence).

Endocytosis was quantified by monitoring cell uptake of human Alexa Fluor 568-conjugated TF (serum transferrin; Thermo Fisher Scientific, Waltham, MA). Monolayer or stratified cultures of HCLE cells were washed once with PBS, and then again with basal K-SFM media. Cells were imaged by phase contrast to ensure that all wells were of equal cell density. Then cells were probed with Alexa Fluor 568-conjugated TF using the manufacturer’s protocol with some modifications. Instead of putting the cells on ice and washing with cold Living Cell Imaging Solution (LCIS), cells were washed with basal K-SFM media. Cells were probed with 10x Alexa Fluor 568-conjugated TF (250 ug/mL) diluted in LCIS for 20 minutes before being washed in cold LCIS. Cells were then imaged with a Keyence BZ-X700 fluorescence microscope (excitation/emission = 532/588 nm). Alexa Fluor 568-conjugated TF uptake was quantified by image J analysis.

Samples were fixed with 4% paraformaldehyde in PBS (pH 7.2) at 4 °C overnight, dehydrated, cleaned and embedded in paraffin. For histology, sections were stained with haematoxylin–eosin. For immunohistochemistry, sections were incubated first with 3% H₂O₂ in ethanol for 30 min to inhibit endogenous peroxidase and then with 10% bovine serum albumin in PBS for 60 min. Sections were treated with 0.01 M citrate buffer (pH 6.0 or 9.0) in a microwave processor (MI-77, Azumaya, Tokyo, Japan) for antigen retrieval. Sections were incubated with primary antibodies (Supplementary Table 2) at 4 °C overnight. Visualisation was achieved using an EnVision + System (Agilent, Santa Clara, CA, USA). Sections were lightly counterstained with haematoxylin. For double-immunofluorescence staining, sections were incubated with primary antibodies (Supplementary Table S2) and then treated with the Opal 3-plex kit (Perkin Elmer). Sections were mounted onto slides using Vectashield Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA, USA). Negative controls utilized isotype-matched antibodies instead of primary antibodies. Images were obtained using a BZ-X700 fluorescence microscope (Keyence, Tokyo, Japan). The number and area of ganglion cells in myenteric ganglia were estimated using Hybrid Cell Count software (Keyence)^{49 (link)–51 (link)}.

Sections of lung tissues were prepared using a cryostat and treated with 10% goat serum for 20 min at room temperature. After washing three times with PBS, the tissues were incubated with rat monoclonal IgG anti‐Crry/p65 (clone 1AF; Becton Dickinson) on ice for 30 min. The tissues were washed three times with PBS, and then incubated with Alexa Fluor 488‐conjugated goat IgG anti‐rat IgG (clone poly4054; Bio Legend) in the dark on ice for 1 h. After washing three times with PBS, the tissues were incubated with Alexa Fluor 594‐conjugated rat monoclonal IgG anti‐mouse CD31 containing Hoechst 33342 solution in the dark on ice for 1 h. The images were captured at 200 × magnification under a BZ‐X700 Fluorescence Microscope (Keyence). To quantify Crry/p65 expression, we measured the brightness of the CD31‐ and Crry/p65‐positive area in the whole section using BZ‐X Analysis software and compared the brightness between vehicle and histone groups.