Sema3e

Mouse models were established as previously described [8 (link)]. In brief, on Days 0, 7, and 14, the AS, EB, and DXM groups were administered with OVA (Sigma Company, USA, A5503-10G) sensitization solution at 200 μL/animal (containing 10 µg of OVA + 1.3 mg of aluminum hydroxide adjuvant (Thermo Fisher Scientific, USA, 77161)) by intraperitoneal injections and challenged with 200, 10, and 10 µg OVA on Days 21–23, respectively. Additionally, the DXM group was treated with 5 mg/kg of DXM (Guangzhou Baiyunshan Tianxin Pharmaceutical Co., Ltd., China, H44022091) by intraperitoneal injection 1 hr before stimulation and measurement of airway reactivity. The NS group was administered equal amount of NS for intraperitoneal sensitization and intranasal stimulation.
In the Sema3E treatment protocol (Figure 1(a)), the AS mouse model was established as described above while challenged i.n. with OVA (200 µg in 25 µL NS), mice were exposed intranasally to Sema3E (5 µg in 25 µL NS) (R&D Systems, Minneapolis, MN., USA, 3239-S3B-025), or NS as a control 1 hr before challenge. Twenty-four hours after the last administration, mice were anesthetized for invasive airway resistance detection and then sacrificed for analysis of airway inflammation, mucus production, and collagen deposition.

For transwell migration assays (BD Biosciences, BIOCOAT® Cell culture inserts, 354578), 2 × 10⁴ breast cancer cells per well were allowed to migrate through 8-μm pores towards control medium (α-MEM + 1% FBS + 1% P/S, referred to as Ctrl), ObCM, or towards α-MEM supplemented with recombinant Semaphorin 3E (Sema3E, R&D Systems, 100-500 ng/ml) for 6 h. Migrated cells were stained using Giemsa’s azur eosin methylene blue solution (Merck, HX8389304). Cells within 4 fields of view of interest were counted using the OsteoMeasure system (Osteometrics) using a × 10 objective (Olympus UPlan Fl 10x/0.30 ∞/).

Eyes of P17 C57BL6/J pups exposed to normoxia or OIR were enucleated, fixed in 4% paraformaldehyde for 1 h at room temperature, and saturated overnight at 4°C in a 30% sucrose solution prior to embedding in OCT compound (TissueTek^®). Sagittal cross-sections of 10 μm was sectioned using a Cryostat (Leica) and permeabilized for 1 h at room temperature. Immunostaining against Sema3E (R&D Systems; 1:100), NeuN (EMD Millipore; 1:100), F4/80 (Abcam; 1:100), or IL-17A (Abcam; 1:200) overnight at 4°C, followed by fluorochrome-conjugated secondary antibody (goat anti-mouse IgG Alexa Fluor 488 and goat anti-rabbit IgG Alexa Fluor 594; Invitrogen) for localization studies according to manufacturers’ recommendations. Nuclei were stained with DAPI (Invitrogen; 1:5000). Cross-sections were visualized using 30× objectives with an IX81 confocal microscope (Olympus), and images were obtained with Fluoview 3.1 software (Olympus).