FOXO1 activation in DLBCL cells induces FOXO1-dependent gene expression, cell cycle arrest, and apoptosis. (A) SYK inhibition induces FOXO1 target gene expression. The heat map shows relative abundance of 40 FOXO1 target genes (GSEA leading edge) in tonic BCR signal-dependent DLBCL cell lines following 24-hour treatment with R406. (B) GSEA enrichment plot of FOXO1 target genes in tonic BCR signal-dependent DLBCL cell lines (DHL4, DHL6, Ly7) treated with DMSO or R406 for 24 hours. Note that the positions of the FOXO1 targets (Vogel et al²³ ) were significantly skewed toward the left end of the sorted list, reflecting their statistically significant induction in R406-treated lines. (C) FOXO1 target gene expression in 4 tonic BCR signal-dependent cell lines following 24-hour treatment with R406 or vehicle, analyzed by qRT-PCR. (P values were determined using 2-sided Gosset’s t test: *P < .05; **P < .01; ***P < .001; ****P < .0001). (D) Protein abundance of key FOXO1 targets, CDKN1B (p27) and BCL2L11 (BIM), in DLBCL cells following 6- to 72-hour treatment with R406 or vehicle, analyzed by western blot. GAPDH served as a loading control. Densitometric quantification of band intensities is provided in supplemental Table 6. (E) FOXO1 nuclear localization induces cell cycle arrest. DHL4 cells were retrovirally transduced with pMIG-FOXO1-WT-IRES-GFP or pMIG-FOXO1-3A-IRES-GFP to express wild-type (FOXO1-WT) or constitutively nuclear FOXO1 mutant (FOXO1-3A). Cell cycle distribution was measured within the GFP⁺ population by Hoechst blue staining and FACS analysis. (F) FOXO1 nuclear localization induces apoptosis. Cells were transduced as in E; 48 hours after transduction, GFP⁺ cells were FACS sorted and after another 60 hours were analyzed by AnnexinV/7-AAD staining. Bar graphs are derived from FACS data.