MSCs are the primary source of IL-6 in the AML BM microenvironment. (A) Coculture of human AML cell lines (HL-60/U937/THP-1 cells) with the human BM stromal cell HS-5 to simulate the BM microenvironment. IL-6 secretion levels of AML and HS-5 cells were measured by enzyme-linked immunosorbent assay (ELISA) after 24 hours of culture (n = 4). (B-C) IL-6 levels in the 24-hour cell culture supernatants measured by ELISA: primary AML cells and MSCs (B) isolated from patients with AML (n = 5); and MLL::AF9 AML cells and mMSCs (C) from MLL::AF9 AML mice (n = 5). (D-E) Validation of Il6 knockout in mouse BM MSCs by polymerase chain reaction (D) and western blot (E). (F) Flowchart for the construction of the Il6_Prrx1^–/– and Il6^fl/fl AML mouse model. (G) FCM analysis of Gr-1 expression in YFP⁺MLL::AF9 cells. (H-I) FCM analysis of the proportion of YFP⁺ AML cells in the PB of mice; representative FCM plots (H); bar chart (I) of YFP⁺ AML cell proportion (n = 5). (J) ELISA analysis of the effect of Il6 deletion in MSCs on IL-6 levels in the BM supernatant of AML mice (n = 5). Data are presented as mean ± standard error of the mean (SEM; panel A) or standard deviation (SD; panels B-C,I-J). Differences were analyzed using ordinary 1-way analysis of variance (ANOVA) with Dunnett multiple comparisons test (panel A; for HS-5 cells), ordinary 1-way ANOVA with Tukey multiple comparisons test (panel I), 2-way ANOVA with Sidak multiple comparisons test (panel A; for AML cells), or 2-tailed unpaired t test (panels B-C,J). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. Co, cocultured; Mono, monocultured; mMSCs, mouse MSCs; Nor, normal healthy mice; ns, not significant.