Iso 1

A normal rat kidney tubule epithelium (NRK-52E) cell line was obtained from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). NRK-52E cells were maintained in Dulbecco's Modified Eagle Medium (Invitrogen, CA, USA) supplemented with 5% fetal bovine serum (FBS, Invitrogen), 100 U/mL penicillin, and 100 μg/mL streptomycin (Invitrogen). The NRK-52E cells were seeded on six-well culture plates to approximately 70% confluence in the complete medium containing 5% FBS for 24 h and then changed to serum-free medium for 24 h before the treatment with 10 μg/mL aristolochic acid I (lot number A5512, Sigma-Aldrich, St. Louis, MO, USA) with or without 50 μmol/L ISO-1 (lot number D00151457, Merck Chemicals, Darmstadt, Germany).

Eosinophil chemotaxis was performed with purified human eosinophils, whereas human PMNL preparations were used to assess the migratory responsiveness of neutrophils. For all experiments, technical triplicates have been performed. Cells were resuspended in assay buffer, pretreated with KPR-6 (20 µM) or ISO-1 (20 µM; Merck) for 30 min at 37 °C, and allowed to migrate towards MIF (3 nM; Peprotech; eosinophils: n = 11, neutrophils: n = 12), IL-8 (10 nM; Immunotools; neutrophils: n = 10) or CCL11 (10 nM; Immunotools; eosinophils: n = 12) for another 60 min at 37 °C in a 48-well micro-Boyden chamber using PVP-free polycarbonate filters with a pore size of 5 µm (eosinophils) or 3 µm (neutrophils) (Sterlitech). Migrated cells were enumerated by flow cytometry on a BD Canto II flow cytometer (acquisition set for 30 s at medium flow rate). Therefore, eosinophils and neutrophils were gated by their forward and side scatter properties and by autofluorescence (Figure S1) [53 (link),54 (link)].