Recombinant human il 17

The human lung adenocarcinoma cell lines A549 (ATCC #CCL-185) and H1299 (ATCC #CRL-1848™) were purchased from ATCC (Manassas, VA, USA) and cultured as previously described21 (link). Recombinant human IL-17 was purchased from PeproTech (Rocky Hill, NJ). The STAT1 inhibitor (fludarabine) was obtained from HISUN Pharmaceutical Co. Ltd. (Zhejiang, China).

When FLS cultures were approximately 90% confluent, cells were incubated with DMEM supplemented with 1% FBS for 4 h. FLS monolayers were wounded with pipette tips and treated for 24 h with recombinant human IL-17 (PeproTech, Cranbury, NJ, USA) and TNF-α (PeproTech). During the blocking assays, the cultures were treated with HIF-2α inhibitor (PT-2385; Abcam) or CD70-blocking antibody (BU69; Abcam). Wound closure was monitored and photographed at 0 and 24 h with an Olympus inverted microscope (magnification 40×; 0.55 numerical aperture dry objective; Tokyo, Japan). To quantify the migrated cells, pictures of the initial wounded monolayers were compared with the corresponding pictures of cells at the end of the incubation.

NHKs were extracted from prepuces obtained from healthy individuals who accepted circumcision. Informed consent was obtained from all donors. Keratinocytes were cultured in the serum-free keratinocyte growth medium (Gibco), and the second- or third-passage keratinocytes were used in all experiments. Cytokine cocktail used for stimulating NHKs was composed of recombinant human TNF-α (50 ng/ml), recombinant human IFN-γ (10 ng/ml), recombinant human IL-17 (25 ng/ml), and recombinant human IL-22 (25 ng/ml) (Peprotech, Rocky Hill City, CT, USA). Peripheral blood mononuclear cells (PBMCs) were obtained from psoriasis patients with informed consent and cultured in Modified Medium RPMI 1640 (HyClone, Logan, UT, USA) with 10% fetal bovine serum (Sijiqing, Hangzhou, China) before subsequent transwell assay. Each experiment was repeatedly performed using the cells at least from three different sources.