Fig 2: Leucine polymer-loaded doxorubicin enhances the chemotherapy efficacy for acute myeloid leukemia cells in vitro. (A) The cell proliferation rate of THP-1 cells treated with phospate-buffered saline (PBS) (control [Ctrl]), 8L6, doxorubicin (DOX), or leucine polymer-loaded doxorubicin (Leu-DOX) as indicated (n=5 independent replicates per group). (B) Representative fluorescence activated cell sorting plots (left) and the quantification (right) of Annexin V+ apoptotic THP-1 cells 48 hours (h) after treatment (n=6 replicates). (C) Representative Images (left) and quantification (right) of the ?-H2AX intensity in THP-1 cells 48 h after indicated treatment (scale bar=1 µm) (n=6 replicates). (D) Representative Images (left) and quantification (right) of DOX fluorescent signal in THP-1 cells at indicated time after treatment with DOX, Leu-DOX, or DOX@polylactic coglycolic acid (PLGA) (scale bar=20 µm). (E) The cell proliferation rate of THP-1 cells treated with PBS (Ctrl), DOX, Leu-DOX, and DOX@PLGA. (n=5 independent replicates per group). (F) Apoptotic THP-1 cells at 72 h after indicated treatment (n=6 independent replicates per group).

Fig 3: The combination of BKM120 and TH588 synergistically induces oxidative DNA damage and apoptosis. a Comet assay of U251 cells following treatment with BKM120, TH588 or the combination for 24 h. H2O2 was used as a control. b Quantification of comet tail moment. Tail moment, calculated as % DNA in the tail multiplied by the tail length. Values represent the average ± SD from three independent experiments (> 25 comets per experiment, **P < 0.01). c Flow cytometry assay of H2AX phosphorylation in U251 cells following treatment with increasing concentrations of TH588 for 24 h. d Quantification of ?-H2AX-positive cells from B (*P < 0.05, **P < 0.01). e Flow cytometric analysis of % apoptotic U251 cells (stained by annexin V/PI) following BKM120, TH588 or combination pretreatment for 24 h. f Quantification of apoptotic cells from f; shown is the average ± SD, n = 3 (*P < 0.05, **P < 0.01). g U251 cells treated with BKM120, TH588 or a combination of both for 48 h, and then subjected with co-staining with annexin V and PI. Cells were observed under a fluorescent microscope and representative photos were given. Scale bars = 0.2 mm. h U251 cells received treatment of BKM120,TH588 or a combination of both for 48 h were immunoblotted with antibodies of cleaved caspase-3 and GAPDH

Fig 4: Runx3 knockdown induces DNA damage and apoptosis in AML cells in vivo. (A) Experimental scheme for getting Runx3 knockdown AML cells induced by MLL-AF9. (B) Representative flow cytometry (FCM) plots and quantitative analysis show the percentage of ?-H2AX+ cells in scramble control and Runx3 knockdown AML cells (n = 5 mice). (C) CLSM images of ?-H2AX expression and quantification of ?-H2AX foci per cell (30 cells in each group) in scramble control and Runx3 knockdown AML cells. Nuclear DNA was counterstained with DAPI. Scale bar = 50 µm. (D) Runx3 knockdown increases apoptosis. Representative FCM plots (left) and statistical results (right) show the percentage of apoptotic cells in scramble control and Runx3 knockdown AML cells. Bone marrow cells were stained for Annexin V and 7-AAD (n = 5 mice).

Fig 5: Training with ß-Glucan Promotes a Beneficial Response to a Secondary Challenge(A) WT mice were injected with ß-glucan or PBS, and after 28 days, they received a secondary challenge with LPS.(B and C) LSK, MPP, and LT-HSC cell numbers (B) and frequency of the same cells in total BM cells (C) at 24 hr after LPS injection (n = 10 mice per group).(D) Representative FACS plots and frequency of ?-H2AX-positive LT-HSCs at 24 hr after LPS injection (n = 10 mice per group, right plots and gray background). The frequency of ?-H2AX-positive LT-HSCs at 28 days after ß-glucan administration in mice not injected with LPS (—) is also shown; ns = 4 and 5, left plots and white background.(E) Experimental protocol for the effect of ß-glucan on the recovery of granulopoiesis after cyclophosphamide administration (4 rounds).(F and G) Total white blood cell (WBC) (F) and granulocyte (Gr1+CD11b+) (G) counts in the peripheral blood (n = 10 mice per group).(H) Experimental protocol for 5-FU administration.(I) Survival curves of 5-FU-treated mice treated with ß-glucan or PBS control (n = 16 mice per group). Comparison of survival curves was performed by log-rank (Mantel-Cox) test, and p value is shown.(J and K) Mice were injected with ß-glucan or PBS, and 7 days later, a single dose of 5-FU was administered.(J) Neutrophil numbers in peripheral blood at different time points after the administration of 5-FU (n = 5 mice per group).(K) Frequency of ?-H2AX-positive LT-HSCs 14 days after 5-FU administration (n = 10 mice per group).Data are presented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.