Article Figures & Data
Figures
- Figure 1.
TPA induces the expression of mda-7/IL-24 and IL-24 delE5 in U937 and HL60 cell lines. U937 and HL60 cells were treated with 20 nmol/L TPA for 0, 24, 48, or 72 hours. A, RT-PCR was done by using primers for mda-7/IL-24 and GAPDH. B, the relative expression levels of mda-7/IL-24 and IL-24 delE5 were analyzed by quantitative real-time RT-PCR in U937 and HL60 cells treated with TPA for 48 hours. Threshold cycle of amplified products, which were amplified by using primers specific to mda-7/IL-24 and IL-24 delE5, is shown. C, the expression of mda-7/IL-24 and IL-24 delE5 protein was assessed by Western blotting. Representative results from 3 independent experiments are shown.
- Figure 2.
RNA interference of the mda-7/IL-24 and IL-24 delE5 genes block TPA-induced monocytic differentiation of U937 and HL60 cells. U937 and HL60 cells were transfected with 100 nmol/L siRNA corresponding to mda-7/IL-24, IL-24 delE5, or nontargeting siRNA (NT). After transfection, differentiation was induced by addition of TPA. A, after 48 hours, the expression of mda-7/IL-24 and IL-24 delE5 protein was assessed by Western blotting, Bands were quantified by scanning densitometry by an LKB Ultrascan XL Laser Densitometer (Kodak Ltd.). B, after 72 hours, monocytic surface markers CD11b, CD14, and CD115 were analyzed by FACS. Results are expressed as the mean ± SD of 3 independent experiments (P < 0.05).
- Figure 3.
Regulation of TPA-induced mda-7/IL-24 and IL-24 delE5 expression and differentiation through the MAPK/ERK pathway in U937 and HL60 cells. U937 and HL60 cells were pretreated with or without the MEK inhibitor U0126 or PD98059 for 30 minutes and subsequently exposed to TPA for 72 hours. A and B, the phosphorylated, total ERK1/2, and mda-7/IL-24 and IL-24 delE5 proteins were determined by Western blotting by using specific antibodies. C, the expression of CD11b, CD14, and CD115 was estimated by FACS analysis. Data are presented as mean ± SD of 3 independent experiments (P < 0.001). D, cellular morphologic changes associated with differentiation were detected with Wright–Giemsa staining. Representative micrographs from 3 independent experiments with similar results are shown. Leica DM4000B microscope, ×40/0.75 HCL PL objective lens, Leica DC500 digital camera. Original magnifications, ×200 (i, iii) and ×1,000 (ii, iv).
- Figure 4.
Ectopic overexpression of mda-7/IL-24 and IL-24 delE5 induces differentiation of leukemic cells. U937 and HL60 cells, and blasts from 3 patients with AML-M5 were transfected with mda-7/IL-24, IL-24 delE5, or the empty vector as a negative control. A, after 48 hours, the expression of mda-7/IL-24 and IL-24 delE5 protein was detected by Western blotting in U937 and HL60 cells. B, after 72 hours, the expression of CD11b, CD14, and CD115 in U937 and HL60 cells was assessed by FACS. Results are expressed as the mean ± SD of 3 independent experiments (P < 0.05). C, after 72 hours, the morphologic changes in blasts from AML patients were analyzed with Wright–Giemsa staining. Arrows indicate the differentiated cells with nuclear condensation and decreased nuclear to cytoplasmic ratio. Leica DM4000B microscope, ×40/0.75 HCL PL objective lens, Leica DC500 digital camera. Original magnifications, ×1,000. D, the expression of CD11b, CD14, and CD115 in blast cells was assessed by FACS.
- Figure 5.
mda-7/IL-24 and IL-24 delE5 have no effect on normal hematopoietic progenitors. Purified CD34+ cells were transfected with mda-7/IL-24 or IL-24 delE5. A, the colonies of CFU-GM, BFU-E, and CFU-GEMM were scored. Results are expressed as the mean ± SE of 3 independent experiments (P > 0.05). B, the expression of CD11b, CD14, and CD115 on cells cultured in liquid medium was detected by FACS on the indicated days. Data are presented as mean ± SD of 3 independent experiments (P < 0.05).
- Figure 6.
mda-7/IL-24 and IL-24 delE5 induce differentiation of leukemic cells by inducing ROS production. U937 and HL60 cells, and blasts from patients with AML-M5 were transfected with mda-7/IL-24 or IL-24 delE5. A, after transfection, cells were cultured for 48 hours, ROS was monitored by FACS. Data are presented as mean ± SD of 3 independent experiments (P < 0.05). B, after transfection, cells were cultured in medium with or without 25 μmol/L NAC for 72 hours. The expression of CD11b, CD14, and CD115 was assessed by FACS. Results are expressed as the mean ± SD of 3 independent experiments from each cell line (P < 0.05). C, after transfection, cells were cultured in medium with or without 10 μmol/L DPI for 48 hours; ROS was monitored by FACS. Data are presented as mean ± SD of 3 independent experiments (P < 0.05). D, NADPH oxidase activity was measured 48 hours after transfection. Data represent the mean ± SE; P < 0.05, compared with vector control. Data are representative of at least 3 different experiments.
Additional Files
Supplementary Data, Yang, et al.
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Volume 10, Issue 4
