Up-regulation of OLFM4 in a subset of AML patients. (A) OLFM4 mRNA expression in human bone marrow (BM) and peripheral blood (PB) neutrophils from normal individuals, as determined by qRT-PCR. OLFM4 expression is shown relative to β-actin mRNA expression. *P < .01. (B) OLFM4 protein expression in BM and PB neutrophils from normal individuals, as determined by Western blot analysis. β-actin expression was used as a loading control. (C) OLFM4 protein expression in subcellular fractions of human BM neutrophils from normal individuals, as determined by Western blot analysis. Expressions of Apaf-1 and MnSOD were used to confirm the identity of the cytosolic and mitochondria fractions, respectively. (D) OLFM4 expression in peripheral blood leukocytes from M1 (n = 7), M2 (n = 10), M3 (n = 12), M4 (n = 10), or M5 (n = 3) subtypes of acute myeloid leukemia (AML) patients and normal individuals (N, n = 10) was analyzed by qRT-PCR. The bar represents the mean level of OLFM4 expression relative to β-actin expression. The values in the M4 group is significantly different from each of the other groups using 1-way ANOVA analysis, followed by Bonferroni multiple comparison test; P < .05. (E) Peripheral blood mononuclear cells from a normal individual (N), and 3 AML patients (P1 [M4 subtype], P2 [M3 subtype], and P3 [M1 subtype]) were immunostained with OLFM4 antibody (1:100) and counterstained with Giemsa. Brown color (arrows) indicates the OLFM4-positive cells.