Upregulation of SIRPα upon differentiation of t(15;17) NB4 cells and induction of cell death following its triggering.

<p>(A) NB4 cells were exposed to 1 µM ATRA and granulocytic differentiation of the cells was examined by cell surface expression of the common myeloid marker, CD11b. (B) SIRPα protein expression, determined by western blotting, is upregulated in ATRA-incubated NB4 cells. β-actin is used as a loading control. (C) Flow cytometric analysis of chSIRPα surface expression is determined by using ED9 mAb in transduced NB4 empty vector and chSIRPα expressing cells. (D) 24 hrs following ED9 (10 µg/ml) incubation, the percentage of cell death in chSIRPα and EV transduced NB4 cells was quantified by APC-Annexin V and PE-7AAD FACS staining. (E) Percentage of apoptosis after exposure to 1 µM ATRA is shown in combination with 10 µg/ml of ED9.</p

SIRPα-derived signal synergizes with different antileukemic drugs.

<p>Inhibition of cell growth is depicted by combination of ED9 mAb (10 µg/ml) with (A) Ara-C and DNR in NB4 cells expressing chSIRPα (B) Ara-C, DNR, VP16, DAC and imatinib in Kasumi-1 cells expressing chSIRPα. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052143#s3" target="_blank">Results</a> are based on means of 3 experiments and are calculated using Calcusyn.</p

SIRPα protein expression in AML cell lines and patients.

<p>Western blot analysis was performed in (A) cell lines and (B) 20 pediatric AML patient samples. β-actin staining was used as loading control. (C) SIRPα expression is quantified relative to β-actin expression.</p

SIRPα upregulation in t(8;21) Kasumi-1 cells following treatment with inhibitors of epigenetic gene silencing.

<p>Kasumi-1 cells were incubated with 1 µM Decitabine, 0.5 mM valproic acid, 1 mM Butyrate and 300 nM Trichostatin. Endogenous SIRPα protein level, determined by Western blotting was upregulated at indicated time points. β-actin staining was used as a loading control.</p

Ligation of chSIRPα induces caspase 3-independent PCD in Kasumi-1 cells.

<p>(A) Flow cytometric analysis of SIRPα expression was performed by using ED9 mAb in stable Kasumi-1 cells expressing chSIRPα and EV. (B) Kasumi-1 chSIRPα and EV cells were incubated with 10 µg/ml ED9 mAb and the percentage of cell death was determined after 24 hrs. Annexin V and 7-AAD FACS staining defined that ligation of chSIRPα resulted in increased cell death in chSIRPα Kasumi-1 cells compared to EV control cells. Data are means ± SD calculated from 3 independent experiments using triplicate samples (*: significant difference <i>p</i><0.05). (C) Kasumi-1 cells expressing chSIRPα or EV were treated with 10 µg/ml ED9 for mentioned time points. Caspase 3 staining shows no cleavage of the p32 subunit. As a positive control for caspase 3 cleavage, human neutrophils (PMN) were incubated at room temperature for 0 and 24 hours.</p