Sb203580

We used transwell chambers (Corning, NY, USA) with a pore size of 8 μm to detect cell migration. In total, 24 h before the experiment, the medium was changed to a serum-free medium, and the cells were starved and placed in a 37°C, 5% CO₂ incubator. The cells were trypsinized and centrifuged. SB203580 (MedChemExpress), Y27632 (MedChemExpress), and LY294002 (MedChemExpress) were diluted in DMEM containing 0.3% BSA at a concentration of 25 nM/ml for SB203580, 10 nM/ml for Y27632, and 10 nM/ml for LY294002. The concentration of the cell suspension was adjusted to 8.0 × 10⁵ cells/ml added to the upper chamber of the Transwell. The intervention group was pretreated by adding the above signal pathway inhibitors in the upper chamber for 30 min, the control group was added with a basal medium in the lower chamber, and the experimental and intervention groups were added with VEGF (40 ng·ml⁻¹) in the lower chamber. After 24 h of incubation at 37°C, the cells that had migrated to the lower surface were fixed with 4% paraformaldehyde for 15 min and stained with 1% crystal violet for 30 min. Then, the migrating cells were photographed with an inverted microscope (×200 magnification) and counted using ImageJ software.

Peripheral blood eosinophils from healthy controls were purified by using an eosinophil isolation kit (Miltenyi Biotec, San Diego, CA, USA, 130-092-010). Eosinophil purity was assayed using flow cytometry and Wright–Giemsa staining (Supplementary Figure 2A). This procedure consistently resulted in a highly purified eosinophil population (95%–99%). These eosinophils (>99% viable by trypan blue exclusion) were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml), streptomycin (0.1 mg/ml), and granulocyte-macrophage colony-stimulating factor (GM-CSF, 50 ng/ml; Novoprotein, Suzhou, China, C003) at 37°C in a humidified atmosphere of 5% CO₂. Then, eosinophils (2 × 10⁵ per well in 200 µl of RPMI) were stimulated in a 96-well plate for 24 or 48 h with or without the addition of the following agents: recombinant TNF-α (50 ng/ml; Novoprotein, Suzhou, China, C008), recombinant IL-5 (50 ng/ml; Novoprotein, Suzhou, China, CI59), 3 μM specific p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 (MedChemExpress, NJ, USA, HY-10256A), and 3 μM SB202474 (a negative analog of SB203580) (MedChemExpress, NJ, USA, HY-112367). At the end of this incubation, eosinophils were harvested and investigated further by using flow cytometry for the expression of CD40 and ICOSL.

Neflamapimod (Nefla) was purchased from MedChemExpress (Monmouth Junction, NJ, USA) and used in the concentration range of 0.001–10 µM. SB203580 was purchased from MedChemExpress and used at a final concentration of 10 µM. Phenylephrine (PE), and 4-aminopyridine (4-AP) purchased from Sigma-Aldrich (St. Louis, MO, USA) were used at a final concentration of 1 µM and 1 mM, respectively. Glibenclamide (10 µM), paxilline (10 µM), nitro-l-arginine (L-NNA, 10 µM), sodium nitroprusside (SNP, 10 µM), indomethacin (indo, 10 µM) and acetylcholine (ACh, 1 µM)) were purchased from Tocris (Minneapolis, MN, USA). 4-AP, SNP, indo and PE were dissolved in distilled water. Nefla, gliben, paxilline, SB203580, L-NNA, and ACh were dissolved in dimethyl sulfoxide (DMSO). The maximum concentration of DMSO in the myograph chamber was < 0.1%, which had no effect on arterial contractility.