Figure 5.
PML/RARα-activated GFI1 is necessary for the maintenance of APL. (A-E) Detailed shRNA-mediated loss-of-function analyses on PML/RARα-activated GFI1. Western blot was performed to validate the knockdown efficiency of shRNAs targeting GFI1 (A). Knockdown of GFI1 induced partial differentiation (B) and apoptosis (C), promoted the G0/G1 phase arrest (D), and decreased the colony-forming capacity (E). The expression of the cell surface differentiation marker CD11b in shGFI1 positively transfected cells was determined by flow cytometry and the morphology was detected by Wright-Giemsa staining. Apoptosis was detected in NB4 cells after transfection of shRNA constructs for 4 days and determined by Annexin V-APC staining. Data represent the mean of 3 replicates ± SD. **P < .001, ***P < .0001. (F) GFI1 function was verified in primary APL blast cells. GFI1 siRNAs or negative control (N.C.) were transfected into blast cells isolated from 2 primary APL patients. (G) The schematic diagram of the in vivo experiment. Murine APL blasts isolated from transplantable APL mice were transduced with the shRNA targeting Gfi1, and then the shRNA-positive cells with ZsGreen1 expression were sorted and transplanted into the recipient FVB/NJ mice. The shRNA empty vector was used as the control. (H) Altered disease onset of APL after Gfi1 knockdown. PML/RARα-positive leukemia cells separated from APL transplantable mice (n = 6) were transduced with shRNA targeting Gfi1 or vector and then transplanted into recipient mice. The statistical significance was calculated by the log-rank (Mantel-Cox) test. **P < .001. (I) Knockdown of Gfi1 in murine APL cells resulted in no obvious splenomegaly in APL transplantable mice. Left panels show the representative images of the spleen. **P < .001. (J) Murine APL blasts were hardly seen in bone marrow of mice upon Gfi1 knockdown, as shown by representative Wright-Giemsa staining of bone marrow smears. (K) The tumor burden in the peripheral blood of mice transplanted with PML/RARα-positive cells with GFI1 knockdown, as compared with those transplanted with cells without GFI1 knockdown. The left panel shows the total white blood cell counts, and the percentage of GFP-positive abnormal promyelocytes is shown in the right panel. **P < .001.

PML/RARα-activated GFI1 is necessary for the maintenance of APL. (A-E) Detailed shRNA-mediated loss-of-function analyses on PML/RARα-activated GFI1. Western blot was performed to validate the knockdown efficiency of shRNAs targeting GFI1 (A). Knockdown of GFI1 induced partial differentiation (B) and apoptosis (C), promoted the G0/G1 phase arrest (D), and decreased the colony-forming capacity (E). The expression of the cell surface differentiation marker CD11b in shGFI1 positively transfected cells was determined by flow cytometry and the morphology was detected by Wright-Giemsa staining. Apoptosis was detected in NB4 cells after transfection of shRNA constructs for 4 days and determined by Annexin V-APC staining. Data represent the mean of 3 replicates ± SD. **P < .001, ***P < .0001. (F) GFI1 function was verified in primary APL blast cells. GFI1 siRNAs or negative control (N.C.) were transfected into blast cells isolated from 2 primary APL patients. (G) The schematic diagram of the in vivo experiment. Murine APL blasts isolated from transplantable APL mice were transduced with the shRNA targeting Gfi1, and then the shRNA-positive cells with ZsGreen1 expression were sorted and transplanted into the recipient FVB/NJ mice. The shRNA empty vector was used as the control. (H) Altered disease onset of APL after Gfi1 knockdown. PML/RARα-positive leukemia cells separated from APL transplantable mice (n = 6) were transduced with shRNA targeting Gfi1 or vector and then transplanted into recipient mice. The statistical significance was calculated by the log-rank (Mantel-Cox) test. **P < .001. (I) Knockdown of Gfi1 in murine APL cells resulted in no obvious splenomegaly in APL transplantable mice. Left panels show the representative images of the spleen. **P < .001. (J) Murine APL blasts were hardly seen in bone marrow of mice upon Gfi1 knockdown, as shown by representative Wright-Giemsa staining of bone marrow smears. (K) The tumor burden in the peripheral blood of mice transplanted with PML/RARα-positive cells with GFI1 knockdown, as compared with those transplanted with cells without GFI1 knockdown. The left panel shows the total white blood cell counts, and the percentage of GFP-positive abnormal promyelocytes is shown in the right panel. **P < .001.

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