Expression of FOXO1 in BCP-ALL. (A) Expression of FOXO1 and FOXO3A proteins and phosphorylation of FOXO1 in BCP-ALL cell lines analyzed by immunoblot. (B) Expression of FOXO1 in PDX ALL analyzed by immunoblot. (C) Relative expression of FOXO1 and FOXO3A mRNA in BCP-ALL cell lines and PDX ALL samples. FOXO1 and FOXO3 mRNA expression was analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The log2 of FOXO1/FOXO3A ratio was calculated as CtFOXO3a-CtFOXO1, where Ct is cycle threshold. Data are shown as mean ± SD (N = 3). (D) Subcellular distribution of FOXO1. After subcellular fractionation, expression of FOXO1 was detected by immunoblot; purity of cytoplasmatic and nuclear fractions was controlled by expression of tubulin B (TUBB) and histone 3 (H3), respectively. (E) Immunofluorescence analysis of FOXO1 localization in BCP-ALL cell lines. Cells were attached to the surface of glass slides by low-speed centrifugation and stained by anti-FOXO1 antibody (green). Nuclei were counterstained with PI (red). Images were acquired using a Zeiss MTB2004 system using Axiovision software (63× oil immersion lens, original magnification ×228). (F) Expression of inducible constitutive-active FOXO1 (FOXO1(3A)ER in BCP-ALL cell lines (F1ER) and wild-type FOXO1 (wtFOXO1). (G) FOXO1 activation is toxic for BCP-ALL cell lines. Pre-BCR+ (018Z and RCH-ACV) and Pre-BCR− (UoCB6) cell lines transduced with FOXO1(3A)ER or with control vector were sorted and treated with 200 nM 4-OHT. After 48 hours, live cells were counted using trypan blue exclusion. The data are presented as percentage of control nontreated cells (mean ± SD). The significance by 2-sided Student t test (N = 3). ****P < .0001.
