Phosphorylated p62

BV2 cells were seeded on 8-well chamber slides (#80841, ibidi) at a density of 2 × 10⁴ per well. The next day, cells were treated as indicated. Then immunofluorescence assays were all performed according to our standard protocol as described before^{47 (link)}. Primary antibodies used were Nrf-2 (1:250; Cell Signaling), Nf-kB (1:250; Cell Signaling), phosphorylated-P62 (1:500; Cell Signaling), and DAPI (1 μg/ml, Thermo Fisher). Images were collected with a Nikon A1+/A1R+ confocal microscope.

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.

BV2 cells were seeded into 6-well plates at a density of 3 × 10⁵ per well. Proteins were collected with M-PER™ Mammalian Protein Extraction Reagent (Thermo Fisher) after 3 h or 24 h incubation with indicated treatment. N2a cells were seeded into 6-well plates at a density of 4 × 10⁵ per well. The next day, N2a cells were exposed to indicated CM for 24 h as mentioned above before protein collection. Western blot was performed as described (4). Primary antibodies used were as follows: COX-2 (1:1000; Cell signaling), iNOS (1:1000; Cell Signaling), α/β tubulin (1:2000; Cell Signaling), Nrf2 (1:500; Cell Signaling), HO-1 (1:4000; Cell Signaling), phosphorylated-P62 (1:2000; Cell Signaling), total P-62 (1:2000; Cell Signaling), cleaved caspase-3 (1:1000; Cell Signaling), caspase-3 (1:1000; Cell Signaling),or β-actin (1:2000; Cell Signaling). A horseradish peroxidase-conjugated secondary antibody (1:10,000; Cell Signaling) and a Supersignal West Femto Maximum Sensitivity Substrate kit (Thermo Fisher) were used for detection. The density of the bands was analyzed using ImageStudioLite software.