(A) Cytokine levels in sera from siCon and siKLF4 cell-inoculated mice were detected by Bio-plex assay. 28 days after tumor cell implantation, sera from tumor-bearing mice (n=3) were collected. Cytokine levels were determined using Bio-plex chemiluminescence assay system (Bio-Rad). * indicates P < 0.05, ** indicates P < 0.0001 versus siCon group. (B) Migratory ability of MDSCs to conditioned medium (CM) derived from siCon and siKLF4 4T1 cells was determined by a transwell migration assay. Recombinant mouse GM-CSF (100 ng/ml), IL-4 (50 ng/ml), and IL-6 (50 ng/ml) were added into the lower chamber. Migrated MDSCs were counted (ten fields per well, triplicate for each experimental groups) 8 h after incubation. Data were presented as mean ± SD from three independent experiments. * indicates P < 0.05 versus siCon group. (C) Maintenance of MDSCs in conditioned media derived from siCon and siKLF4 4T1 cells was evaluated by flow cytometry. Gr-1+ cells were purified from bone marrow of BALB/c mice and incubated with GM-CSF (100 ng/ml) or IL-4 (50 ng/ml) for 6 days. MDSCs were then analyzed by flow cytometry using anti-CD11b and anti-Gr-1 antibodies. A representative result of FACS analysis was shown from three independent experiments. The numbers in bold indicated the mean percentage of MDSCs.