Hepes

A mouse breast cancer cell line (4T1E/M3) was established as previously described [29 (link),30 (link),31 (link)]. The cells were cultured in high-glucose RPMI-1640 medium (Wako, Osaka, Japan) containing 10% fetal bovine serum (FBS, Invitrogen, Thermo Fisher Scientific Inc. Waltham, MA) and 20 mM of HEPES (FUJIFILM Wako Pure Chemical Co. Osaka, Japan) at 37 °C under 5% CO₂. The medium (1.5 mL/dish) was poured into a culture dish attached to the 50 nm SiN–Al holder. The cells (4 × 104; 20 μL/dish) were seeded and cultured at 37 °C under 5% CO₂.
The rat embryonic fibroblasts (REF) cells [26 (link)], kindly provided by Keisuke Ohta (Kurume University School of Medicine), were cultured in a low-glucose D-MEM medium (FUJIFILM Wako Pure Chemical Co.) containing 10% fetal bovine serum (FBS), 20 mM of HEPES, and 4 mM of L-glutamine at 37 °C under 5% CO₂. The cells (4 × 104; 20 μL/dish) were seeded and cultured in 1.5 mL of medium, as described above. The medium was changed after 2–3 days, and the cells formed a sub-confluent or completely confluent monolayer on the SiN membrane in the holder after 3–4 days [22 (link)]. Before immunolabelling, the cells were cultured for 2 h with the medium without fetal bovine serum at 37 °C under 5% CO₂ for starvation to induce autophagy.

OBA9, a human gingival epithelial cell line, was established as previously described^{25 (link),26 (link)} and cultured in HuMedia-KG2 (Kurabo Industries Ltd. Osaka, Japan) containing insulin (10 mg/mL), hEGF (0.1 μg/mL), and hydrocortisone (0.67 mg/mL) at 37 °C under 5% CO₂.
4T1E/M3, a mouse breast cancer cell line, was established as previously described^{35 (link)–37 (link)} and cultured in high-glucose RPMI-1640 medium (Fuji film Wako, Tokyo, Japan) containing 10% fetal bovine serum (GIBCO Thermo Fisher Scientific) and 20 mM HEPES (Fuji film Wako) at 37 °C under 5% CO₂. Cells (4 × 10⁴, 1.5 mL/dish) were cultured on a 50 nm thick SiN film in a hand-made culture dish holder^{22 (link)}.
After 3–4 days of culture, the cells in the holder formed a confluent monolayer on the SiN film in the holder.

Fisher 344 rats (3–4 weeks old) were exposed to general anesthesia. The pulmonary artery was cannulated and perfused with 25 mL PBS containing 50 U/mL heparin (Mochida, Tokyo, Japan) and 1 μg/mL sodium nitroprusside (Sigma-Aldrich), and the lungs were removed. The alveoli were washed twice by injecting and draining PBS from the trachea. The alveoli were then filled with solution A (DMEM/F-12 + 2.5% HEPES (Wako) + 4.5 U/mL elastase (Worthington, Lakewood, NJ, USA) + 0.02 mg/mL DNase I (Sigma-Aldrich)), placed in a flask containing solution A, and shaken in a shaker at 37 °C for 45 min. The peripheral two-thirds of the lungs were then minced, placed in a new flask with solution A, and shaken for 15 min in a shaker at 37 °C. After shaking, a solution containing FBS (DMEM/F-12 + 2.5% HEPES + 50% FBS) was added, and the reaction was stopped by cooling on ice for 5 min. After successive filtration through 100- and 70-µm cell strainers, the lung cells were separated by centrifugation (300× g, 5 min), and the resulting cell pellets were resuspended in DMEM/F-12.