Mle 12

A549 (human type II alveolar epithelial cell) and MLE-12 (mouse type II alveolar epithelial cell) were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). These cells were maintained in Dulbecco’s modified Eagle’s medium for A549 and advanced minimum essential medium for MLE-12 respectively, and supplemented with 2 mM l-glutamine, 10% heat-inactivated FBS (Gibco, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin.

Human alveolar epithelial cell line (A549) was obtained from ATCC (CCL-185). The cells were cultured in DMEM with 10% fetal bovine serum (GIBCO Laboratories, Grand Island, NY), penicillin [100 U/ml] and streptomycin [100 mg/ml] at 37°C in a gas mixture of 5% CO₂ / 95% air in 25 cm² culture flasks (T-25; Corning, Costar). After reaching early confluence the cells were trypsinized and plated for experiments.
Lung epithelial cell lines from mice (MLE-12; CRL-2110) and rat (RLE-6TN; CRL-2300) were purchased from ATCC (Manassas, VA). MLE-12 cells were cultured in DMEM: F-12 medium with insulin (5 ng/ml), transferrin (0.01 mg/ml), sodium selenite (30 nM, hydrocortisone (10 nM), B-estradiol (10 nM), HEPES 10 nM, glutamine and 10% FBS (GIBCO Laboratories, Grand Island, NY). RLE-6TN cells were grown in Ham's F12 medium supplemented with bovine pituitary extract (0.01 mg/ml), insulin (5 ng/ml), insulin-like growth factor (2.5 ng/ml), transferrin (1.25 μg/ml), EGF (2.5 ng/ml), and 10% SFB (GIBCO Laboratories, Grand Island, NY).

The MLE 12 (ATCC CRL-2110) cells were seeded in 96 well plates at 10,000 cells/well in DMEM/F-12 (Gibco, Thermo Fisher Scientific Inc., Waltham, MA, USA), supplemented with 10% (v/v) FBS (Sigma Aldrich, Munich, Germany), 100 IU/mL penicillin, and 100 µg/mL streptomycin (both from Gibco, Thermo Fisher Scientific Inc., Waltham, MA, USA), and cultured overnight in a humidified atmosphere of 5% CO₂, at 37 °C. The cells were then serum-starved for 24 h. For the experiments with CSE, the next day, the cells were treated with different CSE concentrations with or without 10 µM L-NIL for 6 h, and then kept for the additional 16 h after the treatment removal and addition of a fresh culture medium. For experiments with BALF, the cells were treated with BALF diluted 1:10 in a serum-free medium for 24 h. PBS served as a control. The experiment was repeated 18 times in total, by treating the cells in three different experiments with BALF from six different animals per group. Afterwards, the BALF treatment was removed and a new culture medium with 10% (v/v) alamarBlue Cell Viability Reagent (Thermo Fisher Scientific Inc., Waltham, MA, USA) was added. The metabolic activity of the cells was determined after 4 h, in accordance with the manufacturer’s instructions.

The mouse alveolar epithelial cell line (MLE‐12) and mouse monocyte cell line (RAW 264.7) were purchased from ATCC (Manassas). MLE‐12 cells were cultured with F12/DMEM supplemented with 10% foetal bovine serum (FBS; Gibco) in a 6‐cm culture dish; the medium was replaced with serum‐free medium 2 hours before irradiation. Cell irradiation was also performed under Linac with an 8 Gy dosage. RAW 264.7 cells were cultured with DMEM with 10% FBS. All cells were cultured at 37°C in a humidified atmosphere of 95% and 5% CO₂.

Cells were cultured in an incubator at 37°C with 5% CO₂. The A549 and MLE12 cell lines were purchased from ATCC. The immortalized human alveolar epithelial cells A549 were cultured in RPMI 1640 (Gibco, Grand Island, NY, USA) supplemented with 10% bovine calf serum (Sigma-Aldrich, St. Louis, MO, USA). Murine alveolar epithelial cells MLE-12 cells were cultured in DMEM F-12 (Gibco) supplemented with 2% bovine calf serum (Sigma-Aldrich), 1% penicillin and streptomycin (Solarbio), 1% 100 × ITS-G (insulin-transferrin-selenium,Solarbio), 10 nM hydrocortisone (Solarbio), and 10 nM β-estradiol (Solarbio).
To estimate the effect of PTUPB on TGF-β1 (10 ng/mL)-challenged AECs, a series of concentrations of PTUPB (0.1, 1, and 10 μM) were added 1 h before TGF-β1 stimulation. To evaluate the role of Nrf2 in PTUPB-inhibited EMT, ML385 (Cat. No.: HY-100523, MCE) was added to inhibit Nrf2 1 h before the TGF-β1 stimulation.

MLE-12, A549, BEAS-2B and human epithelial kidney 293 T (HEK293T) cell lines were purchased from ATCC. MLE-12, A549 and BEAS-2B cells were cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS, Gibco). HEK293T cells were grown in DMEM supplemented with 10% FBS. Cells were treated with 5-Aza-CdR (0.125, 0.25, 0.5 μM; Sigma), TSA (10, 25, 50 nM; Sigma), or TNF-α (10 ng/mL, 100 ng/mL; Abcam) for 24 h.

The mouse lung epithelial cells MLE-12 (ATCC, Manassas, VA, USA) were cultured in DMEM medium (Gibco, Grand Island, NY, USA) supplemented with 10% FBS (Yeasen, Shanghai, China) and 1% penicillin/streptomycin (Gibco, Grand Island, NY, USA). The cells were incubated in a cell culture chamber at 37 °C with 5% CO₂. MLE-12 cells were seeded at a concentration of 1 × 10⁵/mL in 12-well plates or at a concentration of 2 × 10⁵/mL in 6-well plates. The final concentrations of the drugs used for cell treatment were as follows: MOTS-c (10 μM), ML385 (20 μM), MG132 (20 μM), and CHX (25 μM), respectively [26 (link),27 (link),28 (link),29 (link)].