Dmem high glucose

Mouse fibroblasts (NIH/3T3) (ATCC) were cultured and maintained in Dulbecco’s Modified Eagles Medium (high glucose DMEM, Nacalai Tesque) supplemented with 10% (v/v) Fetal Bovine Serum (FBS, Life Technologies) and 1% (v/v) Penicillin-Streptomycin mixed solution (Nacalai Tesque). Cells were incubated in normal physiological conditions (37°C, 5% CO₂), passaged every three days, and their media was replenished every two days.

We used a mouse macrophage‐like cell line (RAW264.7) and that with a deletion of p62/Sqstm1 (p62‐KO), as described previously (Akiyama et al., 2018). These cells were cultured in high‐glucose DMEM (Nacalai Tesque) containing 10% fetal bovine serum (FBS), penicillin (50 U/ml), and streptomycin (50 mg/ml) in 5% CO₂, and at 95% humidity and 37°C.
To analyze the phagocytic capacity of the RAW264.7 cells, 2.5 × 10⁵ cells were seeded in six‐well plates and incubated for 24 h. To assess phagocytic capacity, DHEA (TCI) in dimethyl sulfoxide (DMSO) was added at 10.0 ng/ml and then incubated for 12 h, after which latex beads in DMSO were added to the cells at 0.66 μl/ml and incubated for 12 h. The cells were then fixed in 4% paraformaldehyde and imaged using a fluorescence microscope. For flow cytometry analysis, the cells in the dish were washed twice and collected using a cell scraper. Next, the collected cells were further washed and centrifuged (4000 rpm for 3 min) twice, and then the fluorescence intensity of the beads in the cells was measured.

All cell lines except for HepG2 except and the gene-edited MEFs^{43 (link)} were obtained from ATCC (Manassas, VA, USA). HepG2 cell identity was estimated to be 100% by the Centre for Translational Research and Diagnostics of the National University of Singapore. All cell lines were cultured in high glucose DMEM (Nacalai, Kyoto, Japan) supplemented with 10% FBS, 2 mM glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin (Thermo Fisher Scientific, Waltham, MA, USA). Primary renal proximal tubule epithelial cells (PRETEC) (ATCC, PCS-400-010) were cultured in the recommended media (ATCC, PCS-400-030 and PCS-400-040) and used within tenfold expansion in cell number. GSK-J4 (12073) and CPI-455 (22127) were obtained from Cayman Chemical (Ann Arbor, MI, USA). Tunicamycin (T-7765) was from Sigma-Aldrich (St Louis, MO, USA). Cycloheximide and actinomycin D were from FUJIFILM Wako (Osaka, Japan). The final DMSO concentration was adjusted to 0.2% in all experiments with inhibitor treatment.

HEK293A cells were cultured at 37 °C in high glucose DMEM (Nacalai Tesque), supplemented with 10% FBS (Sigma). Cells were transfected with plasmid DNA using Polyethylenimine “Max” (Polysciences Inc., Warrington, PA, USA). HEK293A cells were cultured in a 24-well plate, and co-transfected with 0.2 μg each of pTk-(GalRE) × 4-Luc and pCMX-β-galactosidase, along with 0.2 μg of one of the following: pCMX-Gal4 (as a control); pCMX-Gal4-hERRα; pCMX-Gal4-hERRβ; pCMX-Gal4-hERRγ. 24 h after transfection, KUS121, KUS187, esculetin, GSK4716, or GSK5182 were added to the culture medium, and cells were incubated for an additional 24 h. Then, whole cell lysates were prepared, and luciferase and β-galactosidase assays were carried out, as described previously (Saitou et al., 1994 (link), Sasaoka et al., 2014 ). The observed luciferase activities were normalized by the β-galactosidase activities to compensate for different transfection efficiencies (Saitou et al., 1994 (link), Sasaoka et al., 2014 ).

sNF96.2 and HeLa cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and were maintained in high-glucose DMEM (Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum (FBS). We established sNF96.2 cells that stably expressed green fluorescent protein (GFP), and these sNF96.2-GFP cells were maintained in DMEM supplemented with 10% FBS. sNF96.2 cells stably expressing BRAP (G370A), SHP-2 (WT), SHP-2 (G503V), or both SHP-2 (G503V) and either BRAP (WT) or BRAP (G370A), as well as HeLa cells stably expressing SHP-2 (G503V) with or without BRAP (G370A), were cultured in DMEM supplemented with 10% FBS and puromycin (Sigma) at 1 µg/mL or blasticidin (Sigma) at 15 µg/mL.