Figure 7. FC-99 induced monocyte apoptosis and inhibition of their differentiation by up-regulation of let-7a-5p.

All THP-1 monocytes in the following experiments were knocked down using the let-7a inhibitor (50 nM final concentration) for 24 h and were subsequently treated with FC-99 (10 µM) 2 h prior to 24 h incubation with PMA (2.5 ng/mL); NC-inhibitor (50 nM final concentration) was used as a control group. (A) All THP-1 cells were harvested for flow cytometry in order to detect the differentiation markers and the induction of apoptosis, indicating the effect of FC-99 on the expression of the macrophage surface markers, CD11b and CD14, in THP-1 monocytes. (B) Annexin V/PI assay detection of apoptosis in monocytes induced by FC-99.a: NC-inhibitor, b: let-7a-inhibitor, c: NC-inhibitor+FC-99, d: let-7a-inhibitor+FC-99. (C) Western blot analysis of the levels of the apoptotic marker caspase3 and the anti-apoptotic protein BCL-XL. (D) qRT-PCR indicated the mRNA expression of macrophage surface markers, CD11b and CD14, following PMA induction. E. Giemsa staining images were acquired with an optical microscope (original magnification, 200×) and indicated the degree of monocyte differentiation into macrophages. The long arrows represent the undifferentiated monocytes, whereas the short arrows indicate the differentiated macrophages. The results are represented as means ± SEM from four independent experiments. ^*P < 0.05; ^**P < 0.01; ^***P < 0.001 vs. NC-inhibitor group. ^#P < 0.05; ^##P < 0.01; ^###P < 0.005 vs let-7a-5p-inhibitor group. ^&P < 0.05, ^&&P < 0.01, ^&&&P < 0.005, vs. NC-inhibitor+FC-99 group in vitro; ns: no significant difference.