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D. Wu et al.
Wright–Giemsa staining shows that isobavachalcone strikingly reduced the nuclear cytoplasmic ratio (N: C ratio) compared to the control (Figure 4C), suggesting monocytic differentiation. MYC is a main regulator of AML cell differentiation, and DHODH knockout reduces
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the protein level of MYC in HL60 cells (Figure 1H). As expected, after isobavachalcone treatment, there was a significant concentration-dependent loss of MYC expres- sion accompanied by a significant up-regulation of p21 (Figure 4D). Furthermore, MYC protein level was down-
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CDE
FGH
Figure 4. Isobavachalcone induces HL60 cell differentiation. (A) HL60 cells were treated with isobavachalcone for 72 h and CD14 expression was detected by flow cytometry analysis. Right: quantification data of CD14 expression in HL60 cells. (B) HL60 cells were treated with isobavachalcone for 72 h and CD11b expression was detected by flow cytometry analysis. Right: quantification data of CD11b expression in HL60 cells. (C) Morphological changes associated with differentiation of HL60 cells were evidenced by Wright-Giemsa staining in the presence of 30 μM isobavachalcone. (D) HL60 cells were incubated with different concentrations of isobavachalcone for 24 h. After incubation, western blot assay was performed to examine the expression levels of MYC and p21. (E) The expression of MYC was ana- lyzed by western blot 1.5, 3, 6 and 12 h after treatment with 30 μM isobavachalcone. (F) HL60 cells were treated with 30 μM isobavachalcone for 3 h with or without 10 μM MG132. The levels of MYC expression were subsequently examined by western blot analysis. (G) 293T cells were transfected with the MYC-luc reporter plas- mid together with pRSVluc plasmid (as an internal control) and incubated with different concentrations of isobavachalcone for 24 h. (H) HL60 cells were treated with 30 μM isobavachalcone for 24 h, then MYC and p21 gene levels in HL60 cells were examined by reverse transcriptase polymerase chain reaction. Data represent mean ± SD of three independent experiments. A Student t-test was performed, *P<0.05, **P<0.01, ***P<0.001. IBC: isobavachalocone.
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haematologica | 2018; 103(9)