Bleomycin sulfate

Male C57BL/6J mice at 6–7 weeks of age were obtained from Viewsolid Biotech (Beijing, China). Animals were housed at 23 ± 2 °C with a 12 h dark/light cycle. All animals had free access to food and water and were humanely cared for throughout the study. All animals were cared for in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals, and the Experimental Design of this study was approved by the Animal Experimentation Ethics Committee of the Institute of Oceanography, Chinese Academy of Sciences (CTEC-2022-02-01).
Forty-eight male mice were divided equally into four groups, namely (1) negative control group (Neg), (2) BLM group, (3) BLM + Nin (50 mg/kg), and (4) BLM + CCP (200 mg/kg) group. In detail, 5 mg/kg bleomycin sulfate (S1214, Selleck, Japan) was injected into the trachea of the mice, and the mice in the negative control group were injected with an equal amount of normal saline. One day after the BLM injection, the BLM + CCP and BLM + Nin groups were given CCP 200 mg/kg or Nin 50 mg/kg once a day orally, respectively. The BLM and negative control groups were given the same amount of saline. Mice in each group were sacrificed after being over-anesthetized on the 17th day after surgery.

Bleomycin sulfate (purity ≥ 99%) and Actinomycin D (CHD) were purchased from Selleck Chemicals (Houston, TX, USA). Cycloheximide (CHX) was obtained from Sigma (St. Louis, MO, USA). Each compound was dissolved in dimethyl sulfoxide (Sigma) and preserved at − 20 °C. Natalizumab (NTZ) and Anti-Mouse Ly-6G/Ly-6C Antibody (Anti-Ly6G) were procured from MedChemExpress (MCE, Shanghai, China). Human alveolar epithelial cell line A549 were sourced from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). HEK293T-spCas9 cells were purchased from OBiO Technology (Shanghai, China). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 mg/ml streptomycin, and 100 U/ml penicillin at 37 °C under a humidified 5% CO₂ incubator. MLE-12 (iCell-m036) cells were acquired from iCell Bioscience (Shanghai, China) and cultured in specific MLE-12 cell medium (iCell-m036-001b).

Bleomycin sulfate (Selleckchem, Houston, TX, USA, #S1214) was resuspended at 100 μg/mL in dimethyl sulfoxide (DMSO) and used to induce persistent DNA damage in LCLs at 20 μg/mL for two hours. Danazol (Sigma, #D8399) and ML216 BLM helicase inhibitor (Sigma, #SML0661) were resuspended in DMSO at 3 mM and 50 mM, respectively, and used at dilutions noted in the results section.