Fig 1.
Effects of IDR, AraC, and Am80 on cell surface PCA.
Each cell line (EAhy926, HL60, NB4, and U937) was treated with IDR, AraC, or Am80 at 37°C for 8 or 24 h. (A) IDR (8 h), (B) IDR (24 h), (C) AraC (24 h), (D) Am80 (24 h), and (E) IDR + Am80. PCA was measured by normal plasma-based recalcification time. Data are the mean ± SD (n = 6). Significant differences are indicated by (*) when p < 0.05 compared with 0 μM, whereas (**) indicates a significant difference with p < 0.01 compared with 0 μM.
Fig 2.
Effects of IDR and Am80 on expression of surface TF antigen.
Each cell line (EAhy926, HL60, NB4, and U937) was treated with IDR (A) or Am80 (B) at 37°C for 24 h. Cell surface expression of TF antigen was analyzed by flow cytometry. Data are the mean ± SD (n = 6). Significant differences are indicated by (*) for p < 0.05 compared with 0 μM, whereas (**) indicates a significant difference of p < 0.01, and (***) for p < 0.001.
Fig 3.
Effects of IDR and Am80 on expression of surface TM antigen.
Each cell line (EAhy926, HL60, NB4, and U937) was treated with IDR (A) or Am80 (B) at 37°C for 24 h. Cell-surface expression of TM antigen was analyzed by flow cytometry. Data are the mean ± SD (n = 6). Significant differences are indicated by (*) for p < 0.05 compared with 0 μM, whereas (**) indicates a significant difference of p < 0.01, and (***) for p < 0.001.
Fig 4.
Effects of IDR and Am80 on TF mRNA expression.
Each cell line (EAhy926, HL60, NB4, and U937) was treated with IDR or Am80 at 37°C for 4 h. Total RNA was extracted and analyzed by RT-PCR for 28 cycles. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA was used as a loading control. Relative signal intensity was determined. Data are the mean ± SD (n = 6). Significant differences are indicated by (*) when p < 0.05 compared with 0 μM.
Fig 5.
Effects of IDR and Am80 on TM mRNA expression.
Each cell line (EAhy926, HL60, NB4, and U937) was treated with IDR or Am80 at 37°C for 4 h. Total RNA was extracted and analyzed by RT-PCR for 28 cycles. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA was used as a loading control. Relative signal intensity was determined. Data are the mean ± SD (n = 6). Significant differences are indicated by (*) when p < 0.05 compared with 0 μM.
Fig 6.
Effects of IDR and Am80 on cell surface PS exposure.
Each cell line (EAhy926, HL60, NB4, and U937) was treated with IDR (A) or Am80 (B) at 37°C for 24 h. Cell surface expression of PS was analyzed by flow cytometry. PS was detected by FITC-labeled annexin V. Data are the mean ± SD (n = 6). Significant differences are indicated by (*) for p < 0.05 compared with 0 μM, whereas (**) indicates a significant difference of p < 0.01, and (***) for p < 0.001.
Fig 7.
Effects of IDR on cell surface PCA blocked by an anti-TF antibody or annexin V.
(A) To investigate the effect of cell surface TF on PCA induced by 0.2 μM IDR, cell lines were treated with 0.2 μM IDR for 8 h (HL60 cells) or 24 h (EAhy926, NB4, and U937 cells). The cells were then treated with 10 μg/mL mouse monoclonal anti-human TF antibody or the same amount of irrelevant IgG in PBS for 60 min on ice. After washing with PBS, cell surface PCA was assessed as described in Fig 1. Significant differences are indicated by (*) when p < 0.05, whereas (**) indicates a significant difference with p < 0.01. (B) To investigate the effect of cell surface PS exposure in response to 0.2 μM IDR stimulating PCA, after 8 h (HL60 cells) or 24 h (EAhy926, NB4, and U937 cells) of treatment, the cells were treated with 1 μg/mL annexin V in 300 μL annexin V binding buffer at 37°C for 30 min. After incubation, cell surface PCA was assessed as described in Fig 1. Data are the mean ± SD (n = 6). Significant differences are indicated by (*) when p < 0.05, whereas (**) indicates a significant difference with p < 0.01.