FOXO1 mediates toxicity of SYK inhibitor in tonic BCR signal-dependent DLBCL cells. (A) Transcript abundance of FOXO1 target genes, BIM, TRAIL, GADD45α, and CDKN1B (p27Kip1) in cell lines transduced with FOXO1-targeting or control shRNA vectors, assessed by qRT-PCR after 24-hour incubation with R406. (B) SREBP1 expression following incubation with R406 was assessed as described above. (C) Transcript abundance of HRK in DHL4 and Ly7 cell lines transduced with scrambled control shRNA (Scr) or FOXO1-targeting shRNA following 24-hour incubation with R406. P values in A to C were determined using a 2-sided Gosset’s t test: *P < .05; **P < .01; ***P < .001; ****P < .0001. (D) HRK expression was determined after 24-hour treatment with vehicle alone (DMSO), R406, or a combination of SYK and pan-caspase inhibitor (Z-VAD-FMK) by qRT-PCR relative to GAPDH. P values were determined using analysis of variance and post hoc Tukey’s test: ****P < .0001. (E) DHL4 stably transduced with indicated shRNAs were further transduced with the pMIG-HA-DREAM-IRES-GFP vector. GFP+ cells were then FACS sorted and treated with R406 or vehicle alone. After 24 hours of incubation, cells were analyzed for FOXO1 and DREAM expression by immunoblotting. GAPDH served as a loading control. Densitometric quantification of band intensities is provided in supplemental Table 8.
