AML cells pretreated with GSK621 induce murine BMDC maturation in vitro. (A) Experimental schema for coculture experiments. BMDCs were generated over 7 days from fresh bone marrow cells harvested from female C57BL/6 mice. BMDC maturation was measured by flow cytometry after overnight coculture with tumor cells pretreated for 24 hours before mixing. (B) Representative dot plots of the percent activated DCs after overnight coculture with cells pretreated with DMSO, AraC (10 μM), GSK621 (30 μM), or MTO (100 nM), with a DC–to–tumor cell ratio of 1:2. Controls are BMDCs treated overnight with LPS (0.1 μg/mL) or vehicle (PBS). (C) Graphs representing the level of maturation of BMDCs as measured by percentage of MHCII⁺CD80⁺ or MHCII⁺CD86⁺ cells after overnight coculture with C1498 cells pretreated as indicated, with 2 ratios of BMDC to tumor cells; n = 3 replicates (mean ± SD). BMDCs alone were treated with LPS as a positive control to induce activation markers. Ordinary one-way ANOVA with Dunnett multiple comparisons test for multiple comparison to DMSO within each ratio of cocultures, and a t test to compare BMDC cultured without tumor cells, with or without LPS. (D) Coculture experiments using murine AML cells driven by MLL-AF9. Cells were pretreated for 24 hours with GSK621 or vehicle, then mixed overnight with BMDCs in a 1:8 ratio (1 BMDC for 8 AML cells). Percent of live coculture-matured DCs as defined by DAPI-negative, CD11⁺ cells expressing MHCII and CD80, or MHCII and CD86, measured by flow cytometry. Ordinary one-way ANOVA with Šidák multiple comparisons test.