MSC-derived IL-6 increases the active mitochondria in AML cells to resist Ara-C treatment. (A) Experimental layout for assessing mitochondrial function of MLL::AF9 AML cells cocultured with MSCs isolated and purified from mouse BM, followed by treatment with Ara-C or PBS. (B) CCK-8 assay to measure the inhibitory effect of Ara-C on MLL::AF9 AML cells after in vitro coculture (n = 5). (C) Apoptotic cells were determined by FCM analysis of allophycocyanin-annexin V and 7-aminoactinomycin D staining (n = 5). (D-F) FCM analysis of mitochondrial mass (D), mitochondrial membrane potential (E), and mtROS levels (F) in MLL::AF9 AML cells after in vitro coculture (n = 5). (G-H) Laser scanning confocal microscopy (G) and FCM (H) to observe mitochondrial transfer from MSCs to MLL::AF9 AML cells (n = 5; scale bar, 50 μm). (I-J) FCM analysis of mitochondrial transfer from HS-5 (n = 3) (I) or mouse MSCs (n = 5) (J) to AML cells, after pretreatment with anti-human IL-6 (anti–hIL-6) or anti-mouse IL-6 (anti–mIL-6) neutralizing antibodies. Data are presented as mean ± SD (panels B-F,H,J) or SEM (panel I). Differences were analyzed using ordinary 1-way ANOVA with Tukey multiple comparisons test (panels C-F,H), 2-way ANOVA with Sidak multiple comparisons test (panels B,I), or 2-tailed unpaired t test (panel J). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. Co, cocultured; mMSCs, mouse MSCs; Mono, monocultured; ns, not significant.