Figure 4

NMHC activated the NOTCH signaling pathway.

(a) Effect of NMHC on ICN1 generation. HL-60, NB4 and THP-1 cells were treated with indicated doses of NMHC for 24 h. The amounts of ICN1 were detected by immunoblotting and normalized with the internal reference β-actin. (b) Effects of NMHC on the expression of NOTCH target genes. Cells were treated with 1μM NMHC for 6 h and 12 h. HES1 or HEY1 mRNAs were determined by RT-qPCR using 18s rRNA as an internal control. Similarly, FLT3 mRNA was analyzed upon 48 and 72 h NMHC treatments (c). (d) Effect of NMHC, DLL4 and NMHC combined with DLL4 on HES1 expression in NB4. Cells were treated with DLL4 (10 μg/mL), or NMHC (1 μM), or combination of both for 24 h. HES1 mRNA was then analyzed by RT-qPCR. For all quantitative PCR analysis, relative RNA abundance to 18s rRNA is represented as a mean from triplicate wells ± SD. (e) NMHC activated the NOTCH luciferase reporter activity. Luciferase reporter genes harboring NOTCH responsive elements were transfected into 293T cells, NMHC was added 3 h post-transfection for additional 24 h. Relative luciferase reporter activity is represented as a mean from triplicate wells ± SD. (f) NOTCH inactivation renders AML cells less sensitive to NMHC. NB4 cells infected with lentivirus expressing pCDH or pCDH-DNMAML were selected with puromycin (2 μg/mL) for 48 h. Resulting puromycin-resistant cells were subjected to 24 h DMSO or NMHC treatments (1 and 2 μM), stained with Annexin V-APC/PI and analyzed by flow cytometry. Representative data are shown on the left and quantifications relative to untreated cells are on the right. Quantitation is derived from the mean ± SD from three independent experiments. P-values were derived from Student’s t-test (*P < 0.05, **P < 0.01; NMHC treatments vs DMSO treatments).