Figure 2
Figure 2. Depletion of NPM1 induces differentiation of mutant NPM1 expressing AML cells and abolishes OCI-AML3 cell-induced AML in NOD/SCID mice. (A) HL-60, OCI-AML3, and OCI-AML2 cells were transfected with control or NPM1 siRNA and incubated for 72 hours. Then, immunoblot analyses were performed for NPM1, C/EBPα, p53, and p21 on the total cell lysates. The expression of β-actin in the lysates served as the loading control. (B) Top panel, representative light microscopic images of HL-60 and OCI-AML3 cells transfected with control or NPM1 siRNA for 96 hours. Bottom panel, 96 hours post transfection, control and NPM1 siRNA transfected HL-60 and OCI-AML3 cells were cytospun onto glass slides, Wright-Giemsa stained and the percentage of differentiated cells was determined by light microscopy. Values represent the mean of 3 independent experiments ± SEM. (C) OCI-AML3 cells were transfected with control or NPM1 siRNA for 96 hours. At the end of treatment, cells were fixed, permeabilized and stained with DyLight 647-conjugated anti-NPM1 antibody followed by flow cytometry. For determination of CD11b expression, cells were washed with 1X PBS and stained with anti-CD11b antibody. Representative flow histograms of NPM1 and CD11b expression levels are presented. (D) OCI-AML3 cells were transfected with control or NPM1 siRNA for 96 hours. Then, cells were washed and stained with CD11b antibody and the percentages of CD11b positive cells were determined by flow cytometry. Values represent the mean percentage of CD11b positive cells from 3 independent experiments ± SEM. (E) Female NOD/SCID mice were irradiated and then injected in the lateral tail vein with OCI-AML3 sh-NT or OCI-AML3 sh-NPM1 (n = 5 mice per group). The mice were monitored daily for symptoms of leukemia. Survival of the mice in OCI-AML3 sh-NT and OCI-AML3 sh-NPM1 is represented by Kaplan-Meier plot. Median survival of the OCI-AML3 sh-NT leukemia bearing mice was 16 days. (F) When the mice were humanely killed, the spleens were collected and measured. Top panel, the box plot displays the range of spleen size from OCI-AML3 sh-NT and sh-NPM1 leukemia bearing mice. The median spleen size for OCI-AML3 sh-NT mice was 1.6 cm compared with 1.2 cm in the OCI-AML3 sh-NPM1 mice. Bottom panel, representative images of the spleens from 2 OCI-AML3 sh-NT and sh-NPM1 leukemia bearing mice are shown.

Depletion of NPM1 induces differentiation of mutant NPM1 expressing AML cells and abolishes OCI-AML3 cell-induced AML in NOD/SCID mice. (A) HL-60, OCI-AML3, and OCI-AML2 cells were transfected with control or NPM1 siRNA and incubated for 72 hours. Then, immunoblot analyses were performed for NPM1, C/EBPα, p53, and p21 on the total cell lysates. The expression of β-actin in the lysates served as the loading control. (B) Top panel, representative light microscopic images of HL-60 and OCI-AML3 cells transfected with control or NPM1 siRNA for 96 hours. Bottom panel, 96 hours post transfection, control and NPM1 siRNA transfected HL-60 and OCI-AML3 cells were cytospun onto glass slides, Wright-Giemsa stained and the percentage of differentiated cells was determined by light microscopy. Values represent the mean of 3 independent experiments ± SEM. (C) OCI-AML3 cells were transfected with control or NPM1 siRNA for 96 hours. At the end of treatment, cells were fixed, permeabilized and stained with DyLight 647-conjugated anti-NPM1 antibody followed by flow cytometry. For determination of CD11b expression, cells were washed with 1X PBS and stained with anti-CD11b antibody. Representative flow histograms of NPM1 and CD11b expression levels are presented. (D) OCI-AML3 cells were transfected with control or NPM1 siRNA for 96 hours. Then, cells were washed and stained with CD11b antibody and the percentages of CD11b positive cells were determined by flow cytometry. Values represent the mean percentage of CD11b positive cells from 3 independent experiments ± SEM. (E) Female NOD/SCID mice were irradiated and then injected in the lateral tail vein with OCI-AML3 sh-NT or OCI-AML3 sh-NPM1 (n = 5 mice per group). The mice were monitored daily for symptoms of leukemia. Survival of the mice in OCI-AML3 sh-NT and OCI-AML3 sh-NPM1 is represented by Kaplan-Meier plot. Median survival of the OCI-AML3 sh-NT leukemia bearing mice was 16 days. (F) When the mice were humanely killed, the spleens were collected and measured. Top panel, the box plot displays the range of spleen size from OCI-AML3 sh-NT and sh-NPM1 leukemia bearing mice. The median spleen size for OCI-AML3 sh-NT mice was 1.6 cm compared with 1.2 cm in the OCI-AML3 sh-NPM1 mice. Bottom panel, representative images of the spleens from 2 OCI-AML3 sh-NT and sh-NPM1 leukemia bearing mice are shown.

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