Molecular mechanisms by which MSC-derived S100A8 promotes AML progression. (A) Flowchart of rescue experiment with RAGE inhibitor or TLR4 inhibitor added to the cocultivation system. (B) Effect of RAGE inhibitor (FPS-ZM1; 500 nM) or TLR4 inhibitor (resatorvid; 5 μM) treatment on leukemic cell proliferation in the S100A8 overexpression group (n = 4). (C) The cocultivation of MSCs with KASUMI-1 cells after S100A8 overexpression was followed by transcriptome sequencing of the Kasumi-1 cells (n = 3). Gene Ontology analysis of the signaling pathway of Kasumi-1 in the S100A8 overexpression group vs the control group. (D) Gene set enrichment analyses evaluating changes in Kasumi-1 of the S100A8 overexpression group compared to control group. (E) PI3K, Akt expression and its phosphorylation level in Kasumi-1 of the S100A8 overexpression group compared to control group. (F) PI3K, Akt expression, and its phosphorylation level in leukemic cells of S100A8-deficient and control AML mice. (G) Western blot analysis with protein quantification of P-PI3K and p-Akt (n = 4). (H) Effect of PI3K inhibitor (LY294002; 10 μM) treatment on leukemic cells proliferation in the S100A8 overexpression group (n = 4). (I) MFI of ROS in leukemic cells and LSCs in S100A8-deficient and control AML mice (n = 5). (J) MFI of TMRE in leukemic cells and LSCs in S100A8-deficient and control AML mice (n = 5). (K) Representative γ-H2AX images of BM cells of S100A8-deficient and control AML mice (scale bars, 20 μm; n = 5). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. Ctrl, control; FDR, false discovery rate; NES, normalized enrichment score; KO, knockout; q-val, q value.