Keap1

On Day 10, the harvested tongue tissues were stained with 1% toluidine blue dye for 1 min and then washed with 1% acetic acid and PBS to reveal the ulcer sizes on the mouse tongues (n = 3). The tongues were then collected and fixed with 4% paraformaldehyde for 5 days. After dehydration and paraffin embedding, the tissues were sliced into 5 μm thick sections for subsequent histological staining. H&E staining was performed. For immunohistochemical (IHC) staining, primary antibodies including Ki67 (#12202, dilution: 1 : 200, Cell Signaling Technology, USA), nuclear factor erythroid 2-related factor 2 (Nrf2, A0674, dilution: 1 : 200, ABclonal, China), and Kelch-like ECH-associated protein 1 (Keap1, A1820, dilution: 1 : 200, ABclonal, China) were incubated overnight, and secondary antibodies were then incubated for 30 min on the second day, followed by DAB staining. After using hematoxylin, the nuclei were restained. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay was performed using a TUNEL Staining Kit (C1098, Beyotime, China) following the manufacturer's protocol. The sections were observed under a microscope (Olympus, Japan) and photographed. ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used to quantify ulcer sizes, positive cells, and epithelial thickness.

All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee of the Chang Gung Memorial Hospital (approval number: 2018120504). Five-week-old female C57BL/6 mice were treated with the autophagy inhibitor hydroxychloroquine (Sanofi, Paris, France) at a dose 100 mg/kg or a vehicle for five days a week (total treatment duration: five weeks). Thereafter, mice were sacrificed and the paraffin-embedded uterine tissues were sectioned at 4 μm thickness and deparaffinized with xylene. Sections were dehydrated through a series of graded ethanol baths and stained with antibodies raised against ESR1 (Abcam), phosphorylated p62 (Cell signaling), and KEAP1 (Abclonal) in an automated immunohistochemical stainer (Leica bond polymer refine detection kit; Buffalo Grove, IL, USA) according to the manufacturer’s protocol. Hematoxylin was used for counterstaining.

After lysing the cells in a RIPA buffer (150 mM NaCl, 20 mM Tris-Cl pH 7.5, 1% Triton X-100, 1% NP40, 0.1% SDS, and 0.5% deoxycholate) in the presence of proteinase and phosphatase inhibitors (Bionovas, Toronto, ON, Canada), the proteins in the cell lysates were separated through sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred onto nitrocellulose membranes [35 (link)]. The following antibodies were used: KEAP1 (Abclonal, Woburn, MA, USA), p62 (GeneTex, Hsinchu, Taiwan), phosphorylated (Ser349) p62 (Cell Signaling Technology, Danvers, MA, USA), FLAG tag (Sigma-Aldrich), CBP tag (calmodulin-binding peptide, Santa Cruz, for pNTAP expression vector), ubiquitin (Cell Signaling Technology, Danvers, MA, USA), LSD1 (Cell Signaling Technology, Danvers, MA, USA), Mono-Methyl-Histone H3 (Lys4; H3K4me1, Cell Signaling Technology, Danvers, MA, USA), and GAPDH (Santa Cruz Biotechnology, Dallas, TX, USA). The corresponding horseradish peroxidase–conjugated antibodies were obtained from Santa Cruz Biotechnology, and chemiluminescence reagents were acquired from Millipore (Burlington, MA, USA). The signal intensity of autoradiograms was quantified with ImageJ software after normalization to the corresponding GAPDH intensity.