P38 mapk

Epithelial anaplastic human lung cancer CaLu-6 and p22phox^Crispr/Cas9 CaLu-6 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplied with 10% fetal bovine serum (FBS) (Invitrogen Corp., Carlsbad, CA, USA) at 37 °C and 5% CO₂. Cells at 70% of confluence were serum deprived for 24 h and stimulated or not with 10 μM WKYMVm (Primm, Milan, Italy). Unstimulated CaLu-6 cells were used as negative control. In other experiments, CaLu-6 cells were preincubated with the selective FPR2 antagonist WRWWWW (WRW4) (Primm, Milan, Italy) for 15 min at a final concentration of 10 μM, or with 100 μM apocynin, the selective inhibitor of NADPH oxidase (Sigma Chemical, St. Louis, MO, USA), for 2 h, or with 50 μM PD09805, a selective inhibitor of MEK (Sigma), for 90 min, or with 10 μM SB203580, a selective inhibitor of p38MAPK (Sigma), for 1 h. FPR2-unstimulated CaLu-6 cells only preincubated with the appropriate amount of the above-mentioned selective inhibitor were used as a negative control of pretreatments.

PMNs were incubated with viable T. gondii tachyzoites (ratio 1:1, 1:3, or 1:6) for 90 min. To determine if T. gondii-triggered NETs were time-dependent, PMNs were incubated with viable T. gondii tachyzoites (ratio 1:1) for 10, 30, 60, 90, or 120 min. In inhibition tests, cells were pre-treated with 10 μM of the NADPH oxidase inhibitor Diphenyleneiodonium chloride (DPI, Sigma-Aldrich), 10 μM of the p38 MAPK (Sigma-Aldrich) signaling pathway inhibitor SB202190, 100 μM of the Rac1 activation inhibitor NSC23766, or 8.0 nM of the NLRP3 inhibitor MCC950 for 15 min. DNase I (90 U) was also pre-treated with cells for 15 min. Zymosan (1 mg/mL) was used as positive control. Finally, the release of T. gondii-triggered NETs was quantified with Pico Green® (Invitrogen) and the fluorometric reader Infiniti M200® (Tecan, Austria).

ST2 cells (3 × 10⁵ cells/mL) were cultured in a Φ 6 cm plate and stimulated with E. coli-LPS (1 μg/mL), EA (3 μg/mL). Western blotting was performed as described previously by Kudo et al. [35 (link)]. In brief, cell pellets were resuspended in ice-cold lysis buffer. Proteins were separated by SDS-PAGE, electro-blotted onto nitrocellulose membrane, and were visualized by the ECL western blotting detection system (GE Healthcare, UK) (Amersham, Piscataway, NJ, USA). The following antibodies from Cell Signaling Technology were used as primary antibodies: p-NF-κB p65 (#3033; 1 : 1,000), NF-κB p65 (#8242; 1 : 1,000), p-p38mitogen-activated protein kinase (p-p38 MAPK) (#4511; 1 : 1,000), p38 MAPK (#8690; 1 : 1,000), p-SAPK/JNK (#4668; 1 : 1,000), t-SAPK/JNK (#9258; 1 : 1,000), p-IKKα/β (S176/180) (#2697S; 1 : 1,000), IKKβ (#2678; 1 : 1,000), and β-actin (#A2228; 1 : 8,000; Sigma-Aldrich). The following antibody from GeneTex was used as the primary antibody: beta catenin antibody [N1N2-2], N-term (#GTX101435; 1 : 1,000).