Activation of signaling pathways by APRIL. (A) MM1S, RPMI, and OPM2 cells were incubated with APRIL (800 ng/mL) or IGF1 (500 ng/mL) for 30 minutes and analyzed for phospho-AKT expression by Western blotting (top panel). Primary MM cells from 2 cases were treated in the same manner and cell lysates were probed for phospho-AKT expression (bottom panel). (B) Top panels: MM1S, OPM2, and primary MM cells (as indicated) were incubated in medium with or without APRIL (800 ng/mL) for 10 minutes, after which cells were fixed and permeabilized before staining initially with anti–phospho-AKT antibody followed by APC-conjugated goat anti–rabbit IgG. Gray shaded histogram represents control cells; and black unshaded histogram, APRIL-stimulated cells. Bottom panel: MM1S, KMS27PE, and primary MM cells (1 cyclin D1⁺ and 1 D2⁺, with circulating plasma cells) were incubated with or without APRIL for 30 minutes, after which cells were fixed and permeabilized before staining initially with anti-p65 antibody (specific for activated form of p65) followed by APC-conjugated goat anti–mouse IgG. (C) Detection of secreted APRIL by immunohistochemistry using anti-APRIL (C-terminal) in representative BM sections from 2 MM cases (cases 1 [with circulating plasma cells] and 29). Both of these bone marrow biopsies were extensively replaced by CD138⁺ cells (data not shown). Dual staining with anti–BLIMP-1 antibody (iii,vi). (D) Detection of APRIL-producing cells by immunohistochemistry in BM sections from a patient with t(4;14). Sections are stained with anti-APRIL (N-terminal, Stalk) only (in i,ii) and with both anti-CD138 (blue) and anti-APRIL (brown; in panels iii-iv).