American Society of Hematology

Figure 1

$Figure 1. Depletion of NPM1 down-regulates the expression of leukemogenic markers induces G0/G1 accumulation and markedly reduces clonogenic survival of AML cells. (A) Top panel, HL-60 and OCI-AML3 cells were transfected with control siRNA or NPM1 siRNA for 24 hours. After 24 hours, total RNA was extracted and semi-quantitative RT-PCR was performed for WT-NPM1, Mt-NPM1, HOXA9, Meis1, and FLT3. The levels of β-actin mRNA served as the loading control. Bottom panel, quantitative PCR reactions were also performed with SYBR Green to assess NPM1 depletion in the siRNA transfected cells. Relative expression of NPM-1 was normalized to GAPDH. (B) HL-60 and OCI-AML3 cells were transfected with control (−) or NPM1 (+) siRNA and incubated for 48 hours. At the end of incubation, nuclear and cytosolic fractions were isolated and immunoblot analyses were performed for NPM1, HOXA9, Meis1, and FLT3. Expression levels of β-actin and EZH2 served as the loading and fraction controls for the cytosolic and nuclear extracts, respectively. (C) HL-60 and OCI-AML3 cells were transfected with control or NPM1-siRNA and incubated for 96 hours. Then, cells were fixed and stained with propidium iodide and cell cycle status was determined by flow cytometry. Values represent the mean of 3 independent experiments + SEM. (+) indicates G0/G1 values significantly different (P < .05) compared with control siRNA transfected cells. (D) HL-60, OCI-AML3, and U937 cells were transfected with control or NPM1 siRNA and incubated for 48 hours. Then, colony growth in semisolid media was assessed after 8 days. Columns represent the mean of 3 independent experiments; bars represent the SEM.$

Depletion of NPM1 down-regulates the expression of leukemogenic markers induces G0/G1 accumulation and markedly reduces clonogenic survival of AML cells. (A) Top panel, HL-60 and OCI-AML3 cells were transfected with control siRNA or NPM1 siRNA for 24 hours. After 24 hours, total RNA was extracted and semi-quantitative RT-PCR was performed for WT-NPM1, Mt-NPM1, HOXA9, Meis1, and FLT3. The levels of β-actin mRNA served as the loading control. Bottom panel, quantitative PCR reactions were also performed with SYBR Green to assess NPM1 depletion in the siRNA transfected cells. Relative expression of NPM-1 was normalized to GAPDH. (B) HL-60 and OCI-AML3 cells were transfected with control (−) or NPM1 (+) siRNA and incubated for 48 hours. At the end of incubation, nuclear and cytosolic fractions were isolated and immunoblot analyses were performed for NPM1, HOXA9, Meis1, and FLT3. Expression levels of β-actin and EZH2 served as the loading and fraction controls for the cytosolic and nuclear extracts, respectively. (C) HL-60 and OCI-AML3 cells were transfected with control or NPM1-siRNA and incubated for 96 hours. Then, cells were fixed and stained with propidium iodide and cell cycle status was determined by flow cytometry. Values represent the mean of 3 independent experiments + SEM. (+) indicates G0/G1 values significantly different (P < .05) compared with control siRNA transfected cells. (D) HL-60, OCI-AML3, and U937 cells were transfected with control or NPM1 siRNA and incubated for 48 hours. Then, colony growth in semisolid media was assessed after 8 days. Columns represent the mean of 3 independent experiments; bars represent the SEM.

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