Expression of tmTNF-α facilitates the survival of AML cells by modulating multiple signaling pathways. (A-B) SKM-1 cells were treated with 160 μM cytarabine and 50 nM daunorubicin for indicated time points. The apoptosis was detected by staining with propidium iodide (PI) and Annexin-V (B, upper panel), and the double negative cells were gated as surviving leukemia cells after chemotherapy (A, upper panel). tmTNF-α expression was determined in these cells by flow cytometry (A and B, lower panel). Each flow cytogram is representative of 3 independent experiments. The quantitative data represent the mean ± SEM of at least 3 independent experiments. **P < .01; ***P < .001 vs the 0 hours group. (C) Each bar represents the mean ± SEM of the apoptosis rate in control or tmTNF-α shRNA-transfected SKM-1 cells treated by either cytarabine or daunorubicin for 48 hours; **P < .01 vs control shRNA. (D) Each curve represents the mean ± SEM of the cytotoxicity rate of control or tmTNF-α shRNA-transfected SKM-1 cells after treatment with agonistic anti-Fas antibody for 24 hours; *P < .05; ** P < .01; ***P < .001 vs control shRNA-transfection group. (E) Control or tmTNF-α shRNA-transfected SKM-1 cells (1 × 10⁶) were transplanted into recipient NOD-SCID mice to initiate leukemia (n = 5). Recipient mice were euthanized after 6 weeks. At the experimental end point, the percentages of CD45⁺ cells in the murine liver and in palpable tumors were determined by flow cytometry. Data are presented as scatter dot plots with means (horizontal lines); **P < .01 vs the control shRNA-transfected group; ND, FCM assessment was not performed because no mice in the tmTNF-α shRNA group developed any palpable tumors. (F) A heatmap shows gene expression profiling associated with control- or tmTNF-α shRNA-transfected SKM-1 leukemia cells. (G) The most significantly enriched pathways showing differential expression between the 2 groups were identified by DAVID pathway analysis. Each column represents the number of differentially expressed genes between the 2 groups. For differentially expressed genes, the call value of expression had to be P < .05, with a ratio ≥1.5 or ≤0.6667. Both Q and P values are shown. (H) Selected genes that were differentially expressed between the 2 groups were confirmed by qPCR analysis. Values indicate the mean expression ratio comparing the tmTNF-α-knockdown group with the control group.