D5025

For the pulmonary lymphocytes, the right lungs were resuspended with PBS containing 0.1% Collagenase type I (SCR103, Sigma-Aldrich), 0.05% DNase I (D5025, Sigma-Aldrich), and 5% FBS (13011-8611, Tianhang Biotechnology). After incubation at 37°C for 45 min, the tissues were pestled and filtrated with a 200-mesh sieve. The cells were centrifugated and resuspended with PBS, and then treated by Lymphocyte Separation Medium (P8620, Solarbio) as per the manufacturer’s instruction. For the splenic lymphocytes, the spleens were pushed through a 200-mesh sieve to obtain single-cell suspensions, and the suspensions were also treated by Lymphocyte Separation Medium.

Primary striatal medium spiny neuron cultures were prepared as previously described (Kim et al., 2018 (link); Zou et al., 2011 (link)). These cells were utilized given prior work demonstrating the dorsal striatum to be among the largest reservoirs of HIV among post-mortem patients (Nath, 2015 (link)) and for neurons within this region to be selectively vulnerable to Tat-mediated disruption in mice (Schier et al., 2017 (link)). In brief, primary neurons were derived from the striatum of E15-17 C57BL/6J mice. Dissected striata were minced and incubated at 37 °C (5% CO₂) for 30 min with trypsin (2.5 mg/ml; #T4799, Sigma-Aldrich) and DNase (15 μg/ml; D5025, Sigma-Aldrich) in neurobasal media (#21103049; Life Technologies), supplemented with B-27 (#12587010, Life Technologies), L-glutamine (9.5 mM; Life Technologies), glutamate (25 μM; Sigma-Aldrich), and antibiotic/antimycotic mixture (Life Technologies). Cells were centrifuged, triturated, and twice filtered through a 70 μm pore nylon mesh (Greiner Bio-One) and seeded onto poly-L-lysine (#P2636, Sigma Aldrich) coated 24-well plates (7.5 × 10⁴/well) for 7–8 days in supplemented neurobasal media before experimental manipulation.

Leukocytes from decidual tissues were processed, as previously described 28 (link). Briefly, decidual tissues were minced and digested with DNase I (150 µg/mL, D5025; Sigma-Aldrich, Saint Louis, MO, USA), collagenase (1 mg/mL, BS164; Biosharp, China), and hyaluronidase (1 mg/mL, H3506; Sigma-Aldrich, St. Louis, MO, USA) in the Roswell Park Memorial Institute-1640 culture medium. Ten milliliters of this enzyme cocktail was used per 1 g wet weight of tissue, with pulsed digestion of 3 × 20 min at 37 °C with stirring. After each incubation, the tissue was allowed to settle and the supernatant containing the released cells was removed. Finally, the dispersed cells were filtered through a metal sieve and silk, and washed twice with phosphate-buffered saline (PBS, Biosharp, China). The suspensions were loaded onto a Percoll (BS909; Biosharp, China) density gradient to purify the leukocytes. Decidual immune cells with densities of 25-50% were collected.

Tissue samples of ECRSwNP of approximately 1 ml volume were rinsed with normal saline, transferred into 10 ml of DMEM/F12 medium containing 1% penicillin/streptomycin (Hyclone, USA), and digested with 0.1% protease from Streptomyces griseus (9036-06-0, CAS, USA) and 0.1 mg/ml deoxyribonuclease (D5025, Sigma, USA), and incubated at 4°C overnight. Epithelial cells were removed by gently scraping and blow them into single cell to the greatest extent. The medium was then transferred into a 15-ml conical tube, centrifuged at 1500 rpm for 5 min, the supernatant decanted, and the pellet resuspended in DMEM/F12 medium. Primary cells were plated on collagen type IV (Sigma)-coated 12-well culture plates with density of 2×10⁵cells/well for protein extraction and on 24-well culture plates with density of 1×10⁵cells/well for IF, and then cultured at 37°C in 5% CO2 overnight. The medium was then changed to PneumaCultTM-EX plus Basal Medium (05041, CAT, USA) containing 1% penicillin/streptomycin. For EMT induction, recombinant human HMGB1 (P09429, Bio-techne, USA) was added into the plates at different doses (100, 300, 500 ng/ml) for 48 h. Tissues derived from multiple patients and stimulation experiments consisted of a minimum of three independent experiments.

The following antibodies were used in our studies: mouse monoclonal anti-Flag (1:10000, F3165, Sigma-Aldrich), mouse monoclonal anti-HA (1:10 000, H9658, Sigma-Aldrich), rabbit polyclonal anti-53BP1 (IF 1:200, sc-22760, Santa Cruz Biotechnology), rabbit polyclonal anti-MDC1 (1:1000, ab11171, Abcam), rabbit polyclonal anti-BRCA1 (IF 1:100, BS6423, Bioworld), polyclonal anti-γH2AX (IF 1:200, BS4760, Bioworld), rabbit polyclonal anti-Histone H3 (1:10 000, BE3015, EASYBIO), anti-β-Tubulin (1:10 000, BE3212-10, EASYBIO), rabbit polyclonal anti-ESCO2 (1:1000, A301-689A, Bethyl), and mouse monoclonal anti-acetyl-SMC3 K105/106, Clone 21A7 (ChIP 2μg, MABE1073, Millipore). The following reagents were used in our studies: KU55933 (ATM kinase inhibitor, S1092, Selleck Chemicals), NU7441 (DNA-PK inhibitor, S2638, Selleck Chemicals), VE821 (ATR inhibitor, S8007, Selleck Chemicals), DNase I (D5025, Sigma) and bleomycin sulfate (HY-17565, MedChemExpress).

Mixed cortical and hippocampus glial cultures were isolated from P0-P1 pups as described previously^{33 (link)}. Briefly, cortices and hippocampal structures were isolated, and meninges then surgically removed. This cleaned tissue was then placed into a solution of hanks buffered saline solution (HBSS) containing trypsin ethylenediaminetetraacetic acid (EDTA) (1 × final concentration, 59418 C, Sigma) and deoxyribonuclease (DNAse) (1 mg/mL, D5025, Sigma) for 15 minutes, after which the supernatant was collected. Remaining undigested tissue was then subject to a second round of digestion before the supernatants were combined. Cells were plated at a density of 1.25 × 10⁴ cells/mL in culture medium (DMEM containing 20% foetal bovine serum (FBS), 1% penicillin/streptomycin). Media was replaced at days 2, 7 and 14 after which the glial cells formed a monolayer.