Figure 3.
Figure 3. GSTA2 confers proliferation of AML cells through promoting cell cycle advancements. (A) Immunoblot of GSTA2 and GAPDH in MV4-11 cells transduced with lentivirus expressing GSTA2 or control. Cells were incubated with 3 μM of doxycycline for 48 h before being lysed for protein extraction. (B) Growth curves of MV4-11 cells transduced with control or GSTA2-expressing vector (GSTA2 O/E). Cells were cultured in the presence of 3 μM of doxycycline (n = 3). (C) Additive GSTA2 expression-mediated change in the number of cells with S + G2/M-phase DNA content. MV4-11 cells transduced with control or GSTA2-expressing vector (GSTA2 O/E) were cultured in the presence of 3 μM of doxycycline. Forty-eight hours after treatment, cells were harvested and subjected to flow cytometric analysis (n = 3). (D) Frequency of early apoptotic cell death induced by additive GSTA2 expression. MV4-11 cells transduced with control or GSTA2-expressing vector (GSTA2 O/E) were treated as in panel C, and the early apoptotic cells (annexin V+ DAPI−) were scored by flow cytometric analysis (n = 3). (E) Immunoblot of GSTA2 and GAPDH in MV4-11 cells transduced with lentivirus encoding shRNA targeting Luciferase (sh_Luc.) or shRNAs against GSTA2 (sh_GSTA2 1 or sh_GSTA2 2). Cells were incubated with 3 μM of doxycycline for 48 h. (F) Growth curves of MV4-11 cells transduced with lentivirus encoding shRNA targeting Luciferase (sh_Luc.) or shRNAs against GSTA2 (sh_GSTA2 1 or sh_GSTA2 2) with or without simultaneous transduction of shRNA targeting RUNX1 (sh_Rx1_moderate). Cells were cultured in the presence of 3 μM of doxycycline (n = 3). (G) Additive GSTA2 knockdown-mediated change in the number of cells with S + G2/M-phase DNA content were determined in RUNX1 moderately attenuated MV4-11 cells. MV4-11 cells were cultured in the presence of 3 μM of doxycycline as in panel F. Forty-eight hours after treatment, cells were harvested and subjected to flow cytometric analysis (n = 3). (H) Frequency of early apoptotic cell death induced by additive GSTA2 knockdown in RUNX1 moderately attenuated MV4-11 cells. MV4-11 cells were cultured and treated as in panel G, and the early apoptotic cells (annexin V+ DAPI−) were scored by flow cytometric analysis (n = 3). Data are mean ± SEM values. *P < .05, by 2-tailed Student t test. O/E, overexpression.

GSTA2 confers proliferation of AML cells through promoting cell cycle advancements. (A) Immunoblot of GSTA2 and GAPDH in MV4-11 cells transduced with lentivirus expressing GSTA2 or control. Cells were incubated with 3 μM of doxycycline for 48 h before being lysed for protein extraction. (B) Growth curves of MV4-11 cells transduced with control or GSTA2-expressing vector (GSTA2 O/E). Cells were cultured in the presence of 3 μM of doxycycline (n = 3). (C) Additive GSTA2 expression-mediated change in the number of cells with S + G2/M-phase DNA content. MV4-11 cells transduced with control or GSTA2-expressing vector (GSTA2 O/E) were cultured in the presence of 3 μM of doxycycline. Forty-eight hours after treatment, cells were harvested and subjected to flow cytometric analysis (n = 3). (D) Frequency of early apoptotic cell death induced by additive GSTA2 expression. MV4-11 cells transduced with control or GSTA2-expressing vector (GSTA2 O/E) were treated as in panel C, and the early apoptotic cells (annexin V+ DAPI−) were scored by flow cytometric analysis (n = 3). (E) Immunoblot of GSTA2 and GAPDH in MV4-11 cells transduced with lentivirus encoding shRNA targeting Luciferase (sh_Luc.) or shRNAs against GSTA2 (sh_GSTA2 1 or sh_GSTA2 2). Cells were incubated with 3 μM of doxycycline for 48 h. (F) Growth curves of MV4-11 cells transduced with lentivirus encoding shRNA targeting Luciferase (sh_Luc.) or shRNAs against GSTA2 (sh_GSTA2 1 or sh_GSTA2 2) with or without simultaneous transduction of shRNA targeting RUNX1 (sh_Rx1_moderate). Cells were cultured in the presence of 3 μM of doxycycline (n = 3). (G) Additive GSTA2 knockdown-mediated change in the number of cells with S + G2/M-phase DNA content were determined in RUNX1 moderately attenuated MV4-11 cells. MV4-11 cells were cultured in the presence of 3 μM of doxycycline as in panel F. Forty-eight hours after treatment, cells were harvested and subjected to flow cytometric analysis (n = 3). (H) Frequency of early apoptotic cell death induced by additive GSTA2 knockdown in RUNX1 moderately attenuated MV4-11 cells. MV4-11 cells were cultured and treated as in panel G, and the early apoptotic cells (annexin V+ DAPI−) were scored by flow cytometric analysis (n = 3). Data are mean ± SEM values. *P < .05, by 2-tailed Student t test. O/E, overexpression.

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