Human il 6

Two days before transduction, bone marrow cells were harvested from 8 to 12 week old OT-I transgenic mice and cultured at 1.5X107 cells per 10 cm plate in 15 ml DMEM supplemented with 15% heat-inactivated fetal calf serum (FCS), 100 U/mL penicillin, and 100 μg/mL streptomycin, 10mM hepes, 20 ng/ml murine interleukin-3 (IL-3), 50 ng/ml human IL-6 and 50 ng/ml murine stem cell factor (SCF) (R&D Systems). The concentrated virus was transduced into the pre-cultured BMCs. After 48 hours incubation bone marrow cells were collected and washed. Sub lethally irradiated (800 rads) WT B6 recipients were injected IV with 4 × 106 bone marrow cells in PBS. Splenocytes from these BM chimeras were harvested 6–8 weeks post-transplant and were enriched by negative selection using a CD8a⁺ T cell Isolation Kit II (Miltenyi Biotec,). Purity of CD8a⁺ T cells was over 80%. Cells were then stained with anti-CD8 Pac Orange, anti-Thy1.1 PerCP, and anti-ICOS APC and CD8⁺ Thy1.1⁺ ICOS⁺ cells were purified by FACS sorting on a BD FACS Aria. Post-sort ICOS-OT-I T cell populations were over 95% pure.

A cell line of human hepatoma (HepG2 cells) was cultured in Dulbecco's modified eagle medium supplemented with 10% fetal bovine serum and 1% penicillin streptomycin. 1 × 10⁵ cells of HepG2 cells were seeded into a 24-well plate for 24 h. To explore the influence of IL-6 and urea on hepatocyte functions, the cells were incubated with human IL-6 (R&D Systems, Minneapolis, MN, USA) and urea (Sigma-Aldrich, St. Louis, MI, USA) at for 24 h at 50 and 100 ng/mL of IL-6. The total RNA was extracted by RNA easy mini kit (Qiagen, Hilden, Germany). One microgram of total RNA was processed for the reverse transcription with a high-capacity reverse transcription assay (Applied Biosystems, Warrington, UK). Real-time polymerase chain reaction (PCR) was performed on Applied Biosystems 7500 Real-Time PCR System using SYBR® Green PCR Master Mix (Applied Biosystems, Warrington, UK). The primer for CYP3A4, a resemblance human gene with mouse CYP3A11, was CYP3A4-F_5’-CAC AGA TCC CCC TGA AAT TAC GCT TA-3’, CYP3A4-R_5’-AAA ATT CAG GCT CCA CTT ACG GTG-3’. Results were reported as relative quantitation using comparative threshold (delta-delta Ct) method (2^–ΔΔCt) as previously described [27 (link)]. The amount of the target gene in the sample was normalized to β-actin as an endogenous housekeeping gene.

The PMA-differentiated THP-1 cells were treated with javamide-II (0-40 µM) for 10 min, which was then followed by LPS treatment. After 18 h, the media samples were collected to determine the potential effects of javamide-II on the productions of IL-6, IL-1beta and TNF-alpha cytokines. In this cell culture model, LPS was used to stimulate the cells, because the LPS treatment induces macrophage-like THP-1 cells to the M1-like status cells which can produce several inflammatory cytokines including TNF-alpha, IL-1beta and IL-6 [26 (link),27 (link)]. The concentrations of IL-6, IL-1beta and TNF-alpha in the media samples were respectively determined using Human IL-6, Human IL-1beta and TNF-alpha Quantikine ELISA (enzyme-linked immunosorbent assay) Kits from R&D systems (Minneapolis, MN, USA), according to manufacturer’s protocols.

Heparinized bone marrow samples were collected from 6 patients with newly diagnosed Ph⁺ B-ALL (detailed information for these patients are provided in Supplementary Table S2). Mononuclear cells (MNCs) were then separated by density gradient centrifugation using Lymphoprep reagent (Stemcell Technologies). Subsequently, MNCs were cultured in StemSpan basic media (Stemcell Technologies) supplemented with 10 ng/mL human stem cell factor, 10 ng/mL human IL-3, 10 ng/mL human IL-6 (all above cytokines were purchased from R&D Systems), 100 U/mL penicillin, and 100 μg/mL streptomycin (both from BBI Life Sciences). This study was approved by the Institutional Review Board of the Ruijin Hospital affiliated to Shanghai Jiao Tong University School of Medicine. Informed consent for the in vitro drug testing studies was obtained in accordance with the Declaration of Ruijin Hospital affiliated to Shanghai Jiao Tong University School of Medicine.