TRAIL induces NF-κB activation and nuclear translocation in LGL leukemia cells that are blocked by proteasome inhibitors. (A) EMSA demonstrating NF-κB activity in nuclear extracts from T-LGL patient PBMCs (n = 4) and NK-LGL patient PBMCs (n = 3) treated with either saline or rhTRAIL (10 ng/mL) for 2 hours. (B) NF-κB p50 or p65 protein ICC staining as visualized by light microscopy in CD8⁺ cells from an LGL leukemia patient compared with CD8⁺ cells from a normal donor (original magnification ×1000). Cells were treated with control (NTC), TNF-α (positive control, 10 ng/mL), or rhTRAIL (10 ng/mL). Brown staining represents NF-κB p50 or p65 protein. Data are representative of 3 experiments conducted with cells from 3 independent patients. (C) NF-κB p65 protein ICC staining as visualized by light microscopy in CD3⁺/CD8⁺/DcR2⁻ and CD3⁺/CD8⁺/DcR2⁺ cells from an LGL leukemia patient (original magnification ×400). Cells were treated with either vehicle control (NTC) or rhTRAIL (10 ng/mL). Brown staining represents NF-κB p65 protein. Data are representative of experiments conducted with cells from 2 independent patients. (D) NF-κB p50 ELISA of PBMC nuclear protein extracts from T-LGL leukemia patients (n = 4) that were treated with sera from normal donor or sera from LGL patients. Patient cells treated with LGL sera were cotreated with vehicle, TRAIL neutralizing antibody (Neut Ab), or IgG control antibody. (E) EMSA demonstrating NF-κB activity in nuclear extracts from T-LGL patient PBMCs (n = 3) treated with vehicle (DMSO), rhTRAIL (10 ng/mL), or TRAIL (10 ng/mL) plus bortezomib (5 nM). (F) EMSA demonstrating NF-κB activity in nuclear extracts from patient cells from a T-LGL or NK-LGL patient treated with rhTRAIL (10 ng/mL) or rhTRAIL (10 ng/mL) plus increasing doses of ixazomib (0-100 nM). (G) NF-κB p50 or p65 protein ICC staining as visualized by light microscopy in CD8⁺ cells from normal control or LGL leukemia (original magnification ×400). Cells were pretreated with either bortezomib (5 nM) or DMSO for 2 hours followed with the treatment of rhTRAIL (10 ng/mL) or rhTNF-α (10 ng/mL positive control). Brown staining represents NF-κB p50 or p65 protein. Data are representative of 3 experiments conducted with cells from 3 independent patients.