Gsk2656157

Bone marrow cells were differentiated in the presence of recombinant mouse M-CSF (20 ng/ml; PeproTech) in complete medium (RPMI 1640 containing 10 mM glucose, 2 mM L-glutamine, 100 U/mL of penicillin/streptomycin and 10% FBS) for 7 days. Fresh complete medium with M-CSF was supplemented on day 6 prior to use. Day 7 macrophages were washed and variously stimulated with IL-4 (20 ng/ml; PeproTech) or LPS (20 ng/ml; Sigma-Aldrich) plus IFNγ (50 ng/ml; PeproTech) in the absence or presence of 5 μM GSK2656157 (Cayman Chemical), 30 μM CBR-5884 (Cayman Chemical), or 5 μM ISRIB (Cayman Chemical). Macrophages were harvested after 24 h and analyzed by flow cytometry for the expression of M2 or M1 activation. For tumor co-culture experiments, macrophages and tumor cells were collected on day 7. Tumor cells were plated at a density of 5-6 x 10⁵ in a 12-well plate for at least 1 h prior to addition of macrophages. Once tumor cells were attached, 2 x 10⁵ macrophages were added to each well for 72 h. For some experiments, macrophages were cultured with IL-4 in the presence of absence of 1 mM dimethyl-α-KG (dm-KG; Sigma-Aldrich) or 25 μM GSK-J4 (Selleck Chem) for 6 h, and cells were harvested for the further experiments as indicated.