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C. Li et al.
responsive target stability (DARTS) assay and cellular thermal shift assay (CETSA) to clarify whether SP1 is a potential direct binding target of HHT.35,36 Indeed, the DARTS result suggests that HHT directly binds with SP1 protein and confers drug-induced pronase-resistance for SP1 (Figure 3G). Moreover, the CETSA result confirms the direct association of HHT to SP1 and leads to the shift
thermal stability of SP1 protein (Figure 3H). Finally, we showed that depletion of SP1 expression significantly inhibited AML cell growth and suppressed TET1 expres- sion (Figure 3I and J), recapitulating the effects of HHT treatment (Figures 1B and 3C, and Online Supplementary Figure S3A). Thus, by inhibiting the binding of SP1 on TET1 promoter, HHT negatively regulates transcription of
Figure 4. Knockdown of TET1 expression recapitulates effects of homoharringtonine (HHT) treatment in acute myeloid leukemia (AML) cells. (A) Qualitative poly- merase chain reaction (qPCR) analysis of TET1 knockdown efficacy in MONOMAC 6, MA9.3ITD and MA9.3RAS cells. (B) Effects of TET1 knockdown on cell growth/proliferation of these three AML cell lines at different time points [0, 24, 48, 72 and 96 hours (h)]. (C) Effects of TET1 knockdown on apoptosis in MA9.3ITD and MA9.3RAS AML cells. (D) Statistical analysis of apoptosis assay in AML cells from three independent experiments determined by flow cytometry. (E) Effects of TET1 knockdown on cell cycle arrest in AML cells. (F) Statistical analysis of cell cycle assays from three independent experiments determined by flow cytometry. (G) Statistical analysis of cell proportions of CD11b+CD14+ and CD11b–CD14– in TET1 knockdown or control MONOMAC 6 cells. (H) HHT IC50 in MA9.3ITD and MA9.3RAS cells with or without TET1 knockdown. These cells were exposed to HHT for 72 h. *P<0.05; **P<0.01; ***P<0.001; t-test. Error bar, mean±Standard Deviation.
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