Fig. 6

Gilteritinib upregulated NKG2DL expression via the noncanonical NF-κB2/Rel B signalling pathway. A MOLM-13 and MV4-11 cells were treated with IC25 gilteritinib for 24 h followed by real-time PCR. The diagrams showed that gilteritinib elevated the mRNA expression of NF-κB upstream and downstream target genes. B Western blotting on the MOLM-13 and MV4-11 cells pre-treated with IC25 gilteritinib for 24 h. The protein levels of p100, p52, and p65 and the phosphorylation of p100 and p65 in MOLM-13 and MV4-11 cells were significantly increased with gilteritinib treatment. C MOLM-13 and MV4-11 cells were treated with 10 nM TNF-a for 24 h or 2.5uM BAY11-7082 for 12 h following IC25-gilteritinib for 24 h. The gilteritinib-induced upregulation of NKG2DLs in AML cells was similar to that induced by TNF-α but was significantly suppressed by the NF-κB inhibitor BAY 11–7082 at the protein level. D MOLM-13 and MV4-11 cells were transfected with si-NF-kB2 for 72 h and then incubated with IC25-gilteritinib for 24 h. Silencing NF-κB2 downregulated gilteritinib-induced NKG2DL expression in AML cells, indicating that gilteritinib regulated NKG2DL expression via the noncanonical NF-κB2/Rel B signalling pathway. E ChIP analysis showed that NF-κB2 binds to the MICB promoter. F In the absence or presence of 72-h siRNA (siRNA-NC or siRNA- NF-κB2) transfection, MOLM-13 cells were transfected with luciferase reporter vectors (PGL3-vector or PGL3-MICB) using Lipofectamine 3000. MOLM-13 cells were treated with or without IC25-gilteritinib for 24 h, and then the luciferase activity was detected by a dual luciferase assay system. The histogram showed that RLU represents the luciferase activity normalized to pRL-TK luciferase activity for each group. Luciferase assays showed that NF-κB2 was able to activate the transcription of MICB. The data presented are derived from the results of three independent experiments. G, gilteritinib; Con, control; RLUs, relative light units; P-control, pGL3-control vector; ns, not significant. * p < 0˜.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001