CLL exosomes rapidly activate kinases and NF-κB in stromal cells. (A) Phospho-kinase antibody array performed on protein lysates from BM-MSCs and HMEC-1 cells either untreated (Ctrl) or treated for 1 hour with 50 µg/mL CLL exosomes (MEC-1; Exo) (left). Cell lysates were hybridized to membranes containing capture antibodies specific for phosphorylated kinases. Quantification of CLL exosome-induced phosphorylated proteins highlighted by red boxes in left panel (right). Data are reported as fold change (FC) relative to Ctrl. (B) Kinetic of CLL exosome-induced AKT phosphorylation in HS-5 and HMEC-1 cells by immunoblot analysis. ACTB was used as loading control (representative of n = 3). (C) HS-5 and HMEC-1 were preincubated for 30 minutes in the absence (−) or presence (+) of PI3K inhibitor wortmannin (Wort, 100 nM) or MEK inhibitor U0126 (10 µM) before culturing for 5 minutes in the absence (−) or presence (+) of 50 µg/mL CLL exosomes (MEC-1). Expression of AKT and ERK1/2 and their phosphorylated forms (p-AKT and p-ERK1/2) was analyzed by immunoblot. ACTB was used as loading control (representative of n = 3). (D) Immunoblot analysis of IKK-α/β and inhibitory NF-κBα phosphorylation in cell lysates of BM-MSCs and HMEC-1 untreated (Ctrl) or incubated with 50 µg/mL CLL exosomes (MEC-1) for the indicated periods of time. ACTB was used as loading control (representative of n = 3). (E) Representative images of nuclear translocation of p65 in exosome-treated cells.HS-5 and HMEC-1 cells were untreated (Ctrl) or treated with CLL exosomes (Exo; 50 µg/mL) for 1 or 2 hours. After fixation and permeabilization, cells were labeled with anti-p65 antibody (green) and DAPI (blue) and analyzed by confocal microscopy (n = 3). Scale bar, 10 µm.