Bleomycin

For all treatments, rat PTSCs were plated at a density of 5 × 10⁴ cells/well in a 12-well cell culture plate in regular growth media. After the cells were fully attached, the media were replaced with drug-containing growth media with reduced FBS (10%). Specifically, for the bleomycin treatment, cells were treated with 10 μg/ml or 50 μg/ml bleomycin (Millipore) at 37°C with 5% CO₂ for 5 days, and growth media with reduced FBS (10%) were used as control. For the rapamycin treatment, cells were treated with different concentrations of rapamycin (0.1 nM, 1 nM, 10 nM, 25 nM, 50 nM, and 100 nM, Sigma) at 37°C with 5% CO₂ for 3 days, and DMSO was used as control. For the bleomycin and rapamycin treatment, cells were treated with 50 μg/ml bleomycin and 25 nM rapamycin at 37°C with 5% CO₂ for 5 days, with DMSO as control.

HDAC inhibitor (trichostatin A, Sigma, St. Louis, MO) 2 mg/kg was given intraperitoneally 1 h before ventilation based on our present and previous studies that showed 2 mg/kg inhibited HDAC activity [19 (link)]. The mice received a single dosage of 0.075 units of bleomycin in 100 μL of sterile normal saline solution intratracheally (Sigma, St. Louis, MO) and were ventilated 5 days after the administration of bleomycin [8 (link)].

For the generation of deletion mutant of each gene POX04853, POX06496, POX07588, and POX07948, the knockout cassette, containing 5′ and 3′ DNA fragments of the target gene and the G418-resistance gene fragment, was constructed by fusion PCR with specific primer pairs (Supplementary Table S1) according to previously published protocols [25 (link)]. Subsequently, the knockout cassette was directly transformed into fresh protoplasts of strain ΔPoxKu70, the transformants were selected on G418-containing PDA plates, and then knockout candidates were verified by PCR amplification using the special primer pairs (Supplementary Table S1). Similarly, the deletion mutant of PoxMK1 could also be obtained by the above method [23 (link)].
For the creation of a complementary strain of mutant ΔPOXO7948 (ΔPoxMKK1)-, the complementary cassette was used to replace the protease gene PoxPepA, composed of the complete coding region of PoxMKK1, its native promoter and terminator, the bleomycin resistance gene fragment, and the upstream- and downstream-flanking DNA sequence of PoxPepA. The transformants were screened on PDA plate containing 80 μg/mL bleomycin (Sigma-Aldrich, Darmstadt, Germany) and was validated by the special primer pairs (Supplementary Table S1), as previously described by Yan et al. [26 (link)].

Nintedanib was administered once daily through gavage at 30, 60 and 100 mg/kg for 5 days before MV (Boehringer Ingelheim, Biberach, Germany) 21. The mice received a single dosage of 0.075 units of bleomycin in 100 μl of sterile normal saline solution intratracheally (Sigma‐Aldrich, St. Louis, MO, USA) and were ventilated 5 days after the administration of bleomycin 13.

All animal protocols were approved by the Institutional Animal Care and Use Committee of Taipei Veterans General Hospital and accorded with the housing guidelines. Eight-week-old male C57BL/6 mice were obtained from BioLASCO Co., Ltd. (Taipei, Taiwan) and acclimatized for 1 week (5 mice per cage). Normal diet and water offered ad libitum throughout the whole study. All mice randomized to receive PM in 200 μg/20 g, bleomycin (Sigma-Aldrich, USA) in 2 U/kg, bleomycin plus PM, or sterile phosphate-buffered saline (PBS) in control group via intratracheal administration on day 0 (4 mice/group) for one single exposure. PM was sonicated for 30 min, then mixed well with bleomycin in sterile PBS and administrated in bleomycin plus PM group. The mice were placed on the left and right decubitus after treating PM and bleomycin. In treatment test, Reparixin (Tocris, U.K.) was given in 30 mg/kg (in 100 μl saline-diluted dimethyl sulfoxide) by subcutaneous injection at 30 min before administration of bleomycin plus PM, and maintained the same dose on days 1, and 2. Animals were anesthetized by avertin (intraperitoneal injection, 230 mg/kg) and euthanized by overdose of anesthetics. After sacrificing on days 14, the lungs were dissected and fixed in 10% formaldehyde for 24 h. Then the fixative was replaced with PBS for storage of the tissue.