NTS treatment stimulates myeloid colony forming capacity in patient bone marrow cells.

<p>CD34+ cells were cultured in the presence of G-CSF to induce neutrophil differentiation in the absence or presence of 0.5 µM (patient 1) or 1.0 µM (patient 2–3) NTS1 and NTS2. Cells were isolated after 3 days for CFU assays in the presence of G-CSF during 11 days. Data were expressed as the number of CFU-GM (A, upper panel), CFU-G (<b>A, middle panel</b>) and CFU-M (<b>A, lower panel</b>) for each patient and for all patients together (<b>B</b>). Progenitor expansion and terminal differentiation was evaluated after 14 days, Data were expressed as fold induction (<b>C</b>) and the percentage of mature neutrophils and monocytes (<b>D</b>). Error bars represent SEM (between patients). *p = <0.05.</p

Treatment with NTS1 and NTS2 does not affect terminal neutrophil differentiation.

<p>CD34+ cells were cultured in the presence of G-CSF to induce neutrophil differentiation during 17 days. Cells were cultured either in the absence or presence of NTS1 (0.5–5 µM) or NTS2 (0.5–5 µM). After 17 days of neutrophil differentiation was determined by cytospin analysis (<b>A</b>) and FACS analysis or intracellular lactoferrin expression. Data were expressed as the percentage of mature neutrophils (banded or segmented nuclei) (N = 3) (<b>B</b>) the mean lactoferrin expression (MFI) (<b>C</b>) (N = 3), and the percentage of mature monocytes (<b>C</b>). Error bars represent SEM (between experiments). *p = <0.05, **p = <0.01.</p

NTS1 and NTS2 treatment stimulates myelopoiesis and affects C/EBPα and p38MAPK activity.

<p>BALB/c mice were treated with 150 mg/kg 5FU at day 0, followed by treatment with 1 mg/kg NTS1 or NTS2 once a week. Mice were treated 2, 5, 9, 15, or 21 days (3 per group). Mice only treated with 5FU were used as control (2 per group) (<b>A</b>). Bone marrow mononuclear cells were cultured in the presence of rmGM-CSF and rmG-CSF to induce myeloid colony formation during 7 days. Data were expressed as the cumulative number of colonies (<b>B</b>) and the number of CFU-G and CFU-M per femur at day 2, 5, 9, 15 and 21 (<b>C–D</b>). Error bars represent SEM (between mice). *p = <0.05, **p = <0.01. Data are representative for 2 independent experiments. Ba/F3 cells were starved overnight in the presence of 0.5% FCS. Cells were left untreated or treated with NTS1 or NTS2 for 30 minutes, before stimulation with 10% FCS for 15 minutes. CD34+ cells were cultured in the presence of G-CSF for 6 days in the absence or presence of 0.5 or 5.0 µM NTS1 or NTS2. Protein lysates were prepared and Western Blot analysis was performed with an antibody against phosphorylated ERK1/2, phosphorylated p38MAPK and phosphorylated C/EBPα. Antibodies against ERK1/2, p38MAPK, C/EBPα and tubulin were used as controls (<b>E–F</b>). Data are representative for 3 independent experiments.</p