Mab406 sp

VSMCs were pre-treated for 1 h with 50µM Z-VAD.FMK (R&D Systems FMK001) prior to addition of Stau and α-Fas/CHX and incubated for the indicated time points. VSMCs were also pre-treated with 10µM SB203580 (CELL, SM32) and 25µM SP600125 (Sigma, S5567). Recombinant proteins were purchased from Peprotech. Cells were treated with 100 ng/ml IL-6 (216 − 16) and GM-CSF (315-03) or 50 ng/ml of each for combined treatment. Neutralization of IL-6 and GM-CSF in conditioned medium was achieved with 0.75 µg/ml of anti-GM-CSF (R&D MAB415-SP) and anti-IL-6 (R&D MAB406-SP). Inhibition of transcription was performed with 2 µg/ml of Actinomycin-D (NovusBio, NB1229).

For the neutrophils and MSCs coculture assay, 1 × 10⁵ MSCs were cultured in 24-well plate in a volume of 500 μl α-MEM with 15% FBS. After 24h of culture, the MSCs were cultured with 1 × 10⁶Rheb1^fl/fl or Rheb1^Δ/Δ neutrophils using cell culture inserts (FALCON, 353095, United States). After 24 h of coculture, the MSCs were harvested, and the relative expression of stem cell factor (SCF) was measured. For the IL-6 neutralization experiment, IL-6 antibody (R&D, MAB406-SP, United States) was added to the coculture system at 10 ng/ml. After 24 h of coculture, MSCs were harvested, and the relative expression of SCF was measured. All cells were incubated at 37°C in a 5% CO₂ incubator.

BM cells were isolated from the tibias, femurs and ilia of 8-week-old B6. SJL mice. WT LK⁺ cells were sorted with a c-Kit (CD117) Microbead Kit (MACS, 130-091-224, German) and a Lineage Cell Depletion Kit (MACS, 130-090-858, German) according to the manufacturer’s protocol. 5×10⁴ MSCs were cultured in 24-well plate in a volume of 800 μL α-MEM with 15% FBS and treat with rapamycin or ethanol. After 24 h of culture, the MSCs were cocultured with 2×10⁵ LK⁺ cells. After 24 h of coculture, the MSCs were harvested. The LK⁺ cells were analyzed for the percentage and absolute number of myeloid cells by flow cytometry. For the G-CSF neutralization experiment, G-CSF antibody (R&D, MAB406-SP, United States) was added to the coculture system at 2 nM. After 24 h of coculture. The LK⁺ cells were analyzed for the percentage and absolute number of myeloid cells by flow cytometry. All cells were incubated at 37°C in a 5% CO₂ incubator.