Interleukin 6 (il 6)

The recombinant proteins of TNF-α (catalog^#1130-01), IL-1β (catalog^#1110-01B), and IL-6 (catalog^#1110-06) were purchased from the GOLDBIO.COM, USA. Based on the in silico analysis of this study, the designed peptide (KCF18), a truncated peptide (SEM18) derived from TNFR1, a mutant peptide (mKCF18), and a random peptide for negative control (CF25) were chemically synthesized by GeneMark (GMbiolab Co., Ltd., Taiwan) with a solid phase methodology for in vitro and in vivo experimental confirmation.

Wild-type or HMGN1-OE cells stably infected with the oncogenes indicated were seeded in methylcellulose media (Methocult M3234, Stem Cell Technologies), supplemented with IL-6 (10 ng/ml), SCF (10 ng/ml), and IL-3 (6 ng/ml) (GoldBio) at 2 × 10⁵ cells/ml and at 5 × 10⁴ cells/ml in subsequent passages. Colonies were manually counted at 7 days, pooled, and replated. For long-term culture initiating colony (LTC-IC) assays, leukemic splenocytes from three different animals were plated on MS5 feeder cells for three weeks in RPMI media enriched with IL-6 (10 ng/ml), SCF (10 ng/ml), and IL-3 (6 ng/ml). Cells were counted and 10,000, 20,000, or 50,000 GFP+ cells were seeded in methylcellulose media (Methocult M3234) supplemented with SCF (10 ng/ml), IL-3 (6 ng/ml), and IL-6 (10 ng/ml) in 35 mm dishes. One week later, the number of colonies and cells was determined.

To perform the test of monocyte adherence to endothelial cells monolayers, the HMEC-1 cell monolayers were cultivated on 24-well plates under serum-starvation condition prior to the monocyte binding assay. The peptides were added to the HMEC-1 cells for one hour prior to the activation by 20 ng/ml TNF-α (Sigma, USA), IL-1β (GOLDBIO, USA) and IL-6 (GOLDBIO, USA) for 18 hours. The THP-1 monocytes pre-labeled with 5 mM Calcein-AM (Invitrogen, USA) for 30 minutes in RPMI 1640. After the monolayers were washed twice to eliminate TNF-α, IL-1β and IL-6, the pre-labeled THP-1 cells (5 × 10⁵ cells/well) were incubated with HMEC-1 and cultivated for 30 minutes at 37 °C. The non-stuck THP-1 cells were removed from monolayers by Hank’s balanced salt solution five times. The adhered monocytes were detected and photographed (five images per well) by fluorescent microscopy and the amount of stuck THP-1 cells were counted by the software AlphaImager 2200 (Alpha Innotech, USA).