FIGURE 6.

S100A9 mediates the effect of ATF3 on G-MDSCs. A, expression of S100A8/A9 in splenic MDSCs was determined by qRT-PCR (upper) and Western blotting (below). B, BM cells were infected with lentivirus expressing ATF3 or vector (with GFP tag); gene expression in GFP⁺ cells was evaluated by qRT-PCR (upper) and Western blotting (below). C, levels of S100A8/A9 protein in the serum from the indicated animals were measured by ELISA. D, phosphorylation of STAT3 and STAT5 in splenic MDSCs was measured by flow cytometry (left), the STAT6 phosphorylation was evaluated by Western blotting (right). MFI, mean fluorescence intensity. E, scheme of S100A8/A9 promoters with potential ATF3-binding sites as indicated. S100A8: site 1, −416 to −425 bp, and site 2, −954 to −961 bp; S100A9: site 1, −506 to −515 bp, and site 2, −1324 to −1331 bp. Overexpressed ATF3 lentivirus were infected with WT BM cells for 3 days, and then cells were fixed in 1% paraformaldehyde for ChIP assay. The presence of S100A8/A9 promoters harboring the ATF3-binding sites was determined by qRT-PCR. Data were normalized against input and presented as fold increase over IgG control. F, S100A8/A9-Luc activity, 32D cell transfected with pS100A8/Luc or pS100A9/Luc plasmid and then were infected with overexpressed ATF3 lentivirus. Luciferase activity was measured using the Dual-Luciferase Reporter Assay (Promega). G, BM cells were cultured with GM-CSF and IL6 in presence or absence of neutralizing antibodies against S100A8 or S100A9, and anti-IgG was used as control. The percentages of the indicated populations were analyzed by flow cytometry. A–C and E–G, results were mean ± S.E. from three independent experiments; Western blots in A, B, and D are representative data from three independent experiments, each sample was pooled from three mice. *, p < 0.05; **, p < 0.01, unpaired t tests were used. ns, no significance; RLU, relative luciferase units; Mac, macrophage.