Cxcl2

Neutrophil migration was assessed using 24-well microchambers and polycarbonate filters (5 μm pore size) (Corning, NY, USA) as described previously^{40 (link),64 (link)}. In brief, CD45⁺ CD11b⁺ Ly6G⁺ neutrophils sorted from mouse bone marrow cells were placed in the upper wells (1 × 10⁵ cells/well) of Transwell chambers, and 600 µL of RPMI 1640 medium with or without 100 ng/ml CXCL2 (BioLegend) were added to the lower wells. For migration assays, cells were incubated for 60 min at 37 °C. After the incubation, cells that migrated to the bottom part of the membrane and the lower wells was stained with Hoechst 33,342. The numbers of cells per field were counted in 10 randomly selected visual fields under a microscope (BZ-X810, Keyence, Osaka, Japan), and the mean estimate for individual samples was calculated.

Chemotaxis assay was performed on freshly isolated WT and Nrf2 KO PMNs using transwell migration assay, as previously described [37 (link)]. Briefly, PMNs (1 × 10⁶ cells) were added to the upper chamber of Transwell filters (3 μm pore diameter, Costar). These chambers were placed in 24-well cell culture plates containing 600 μL assay buffer without chemoattractant or with N-Formylmethionyl-leucyl-phenylalanine (fMLP, 1 μM) (Sigma-Aldrich), increasing concentrations of CXCL2 (0.5–200 nM) and increasing concentrations of CXCL12 (50–400 nM) (both from BioLegend). In some experiments, cells were preincubated with AMD3100 octahydrochloride 50 nM (Sigma-Aldrich) for 30 min before being placed in the upper Transwell chamber to confirm the specificity of CXCR4-dependent migration. Chambers were then incubated for 60 min at 37°C with 5% CO2 and the cells that had migrated to the bottom chamber were recovered and stained with antibodies against Ly6G (PercP. Cy5.5) and CD11b (FITC) (both from BioLegend) for flow cytometry analysis (FACS Calibur). Chemotactic indexes were then calculated by dividing the number of PMNs counted in chemokine-stimulated wells by the number of PMNs counted in filter-free wells (input well without any chemokine).

The CyQUANT proliferation assay with HUVEC (Promocell, Heidelberg, Germany) was performed according to the manufacturer’s protocol (CyQUANT Direct Cell Proliferation Assay, Life Technologies, Carlsbad, CA, USA).
16,000 HUVEC cells were seeded per well in a 96-well flat-bottomed µ-clear black plate (Greiner Bio-One, Kremsmünster, Austria) in ECGM2 media (Promocell, Heidelberg, Germany) and grew for 24 h. The cells were starved overnight in ECGM2 medium with 0.1% FCS but without growth factors. Afterwards, the recombinant human proteins VEGF₁₆₅ (BioLegend, 583704), CXCL2 (BioLegend, 582004), IL8 (BioLegend, 574204) were added in starvation medium to the cells at different concentrations. After 24 h CyQUANT reagent was added to the cells for 1 h.
Images of cells were taken and analyzed by ImageJ 1.53c, or the relative fluorescence intensity was measured using a multi-well spectrophotometer (Tecan, Männedorf, Switzerland) at the excitation wavelength of 485 nm and the emission wavelength of 525 nm.