PRMT5 inhibition activates the FOXO1-dependent transcription program in MCL. (A) Immunoprecipitation of AKT in CCMCL1 and Z-138 cells shows the decrease in interaction between AKT and FOXO1 with treatment of 100 or 150 nM, respectively, of PRT382 for 6 days. (B) Immunofluorescence of CCMCL1 and Z-138 cell lines looking at the localization of FOXO1 after 72 hours of treatment with 100nM of PRT382 or dimethyl sulfoxide (DMSO). Cells are stained with FOXO1 primary antibody and alexafluor488-conjugated donkey anti-rabbit secondary. Cells were also stained with 4′,6-diamidino-2-phenylindole (DAPI). Images taken on an EVOS FL Cell Auto Imaging system at 40× original magnification. The number of cells with FOXO1 enriched in the nucleus was quantified and graphed. Scale bar, 10 μM. (C) Traces of ChIP sequencing performed on FOXO1 in CCMCL1 cells treated for 48 hours with 100 nM of PRT382 or DMSO as a control. Selected BCL-2 family proteins are shown here. (D) FOXO1 consensus sequence was confirmed in the promoter sequences of BAX upstream of the gene body. (E) FOXO1 ChIP qPCR of BAX showing significant enrichment in Z-138, Maver-1, and SP53 as well as a trend in CCMCL1, Jeko, and UPN-1 after 72 hours of PRT382 treatment. A student t test was performed to show significance for panels B,E. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. Error bars show standard deviation of the data.