Figure 2.
Human AML cells are sensitive to GADD45g overexpression. (A) qRT-PCR evaluation of GADD45g expression in AML cells with or without Dox-induced GADD45g overexpression and representative western blot in THP-1 cells. Effects of Dox-induced GADD45g expression on cell proliferation (B), colony formation (C), and apoptosis (D) in THP-1 cells as determined by cell counting, colony-forming assay, and flow cytometric analysis of Annexin V and 7AAD staining, respectively. (E) Primary BMMNCs from patients with AML (n = 5) and cord blood mononuclear cells from healthy donors (n = 5) were transfected with human GADD45g construct, and the percentage of apoptosis cells in green fluorescent protein (GFP)-positive cells were determined by fluorescence-activated cell sorting (FACS) analysis of Annexin V and 7AAD staining. (F) Colony formation of primary BM CD34+ from patients with AML (n = 5) and CB CD34+ cells from healthy donors (n = 3) after lentivirally transduced with GADD45g or empty vector control. (G) Primary BMMNCs from patients with AML (n = 5) were transfected with GADD45g small hairpin RNA (shGG) or scramble small hairpin RNA (shCtrl), and the percentage of apoptosis cells in GFP-positive cells were determined by FACS analysis of Annexin V and 7AAD staining. (H) Effects of Dox-induced GADD45g expression on cell differentiation of THP-1 cells, as determined by flow cytometric analysis of side-scatter profiles (SSC) and the expression of CD11b and CD14, and representative May-Grünwald-Giemsa staining in THP-1 cells with or without Dox treatment. Original magnification, ×400. (I) Effects of GADD45g overexpression on cell differentiation of primary BM CD34+ from patients with AML (n = 3) and CB CD34+ cells from healthy donors (n = 3), as determined by flow cytometric analysis of the expression of CD11b and CD14. THP-1 cells with GADD45g overexpression (J) or knockdown (K) were exposed to the indicated concentrations of daunorubicin (DNR) or VP-16 for 24 hours, and the percentage of apoptosis cells was determined by FACS analysis of Annexin V and 7AAD staining. Data are presented as mean ± SD of ≥3 independent experiments, and comparisons were evaluated by using the 2-tailed Student t test. *P < .05, **P < .01, ***P < .001. MFI, mean fluorescence intensity; NS, not significant.

Human AML cells are sensitive to GADD45g overexpression. (A) qRT-PCR evaluation of GADD45g expression in AML cells with or without Dox-induced GADD45g overexpression and representative western blot in THP-1 cells. Effects of Dox-induced GADD45g expression on cell proliferation (B), colony formation (C), and apoptosis (D) in THP-1 cells as determined by cell counting, colony-forming assay, and flow cytometric analysis of Annexin V and 7AAD staining, respectively. (E) Primary BMMNCs from patients with AML (n = 5) and cord blood mononuclear cells from healthy donors (n = 5) were transfected with human GADD45g construct, and the percentage of apoptosis cells in green fluorescent protein (GFP)-positive cells were determined by fluorescence-activated cell sorting (FACS) analysis of Annexin V and 7AAD staining. (F) Colony formation of primary BM CD34+ from patients with AML (n = 5) and CB CD34+ cells from healthy donors (n = 3) after lentivirally transduced with GADD45g or empty vector control. (G) Primary BMMNCs from patients with AML (n = 5) were transfected with GADD45g small hairpin RNA (shGG) or scramble small hairpin RNA (shCtrl), and the percentage of apoptosis cells in GFP-positive cells were determined by FACS analysis of Annexin V and 7AAD staining. (H) Effects of Dox-induced GADD45g expression on cell differentiation of THP-1 cells, as determined by flow cytometric analysis of side-scatter profiles (SSC) and the expression of CD11b and CD14, and representative May-Grünwald-Giemsa staining in THP-1 cells with or without Dox treatment. Original magnification, ×400. (I) Effects of GADD45g overexpression on cell differentiation of primary BM CD34+ from patients with AML (n = 3) and CB CD34+ cells from healthy donors (n = 3), as determined by flow cytometric analysis of the expression of CD11b and CD14. THP-1 cells with GADD45g overexpression (J) or knockdown (K) were exposed to the indicated concentrations of daunorubicin (DNR) or VP-16 for 24 hours, and the percentage of apoptosis cells was determined by FACS analysis of Annexin V and 7AAD staining. Data are presented as mean ± SD of ≥3 independent experiments, and comparisons were evaluated by using the 2-tailed Student t test. *P < .05, **P < .01, ***P < .001. MFI, mean fluorescence intensity; NS, not significant.

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