Ibrutinib suppresses BCR- and coculture-stimulated signaling and cytokine and chemokine production of MCL cells. (A) Mino cells pretreated with vehicle or ibrutinib (10, 100, or 1000 nM) were cultured alone (with anti-IgM stimulation) or in coculture with murine M2-10B4 stromal cells. M2-10B4 cells alone were pretreated with either vehicle or 100 nM ibrutinib (M2 only). Mino cells in coculture, Mino cells alone, or M2-10B4 cells alone were collected after 48 hours, lysed, and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blot analysis of pY223 BTK, pY1217 PLCγ2, pT202/Y204 ERK, pS473 AKT, and pT183/Y185 JNK. The phosphoblot was scanned, and signals were quantified (right panel); pBTK, pPLCγ2, pERK, pAKT, and pJNK signals were normalized to BTK, PLCγ2, ERK, AKT, and JNK total protein, respectively. Signals from each treatment were compared with vehicle-treated Mino cells. (B) Conditioned media from Mino cells only without stimulation, Mino cells stimulated with anti-IgM, or Mino cells in coculture with M2-10B4 or M2-10B4 alone were collected after 48 hours and analyzed for human cytokines and chemokines (IL-10, CCL22, CCL3, CCL4, TNF-α, CCL17, and CCL21). Note media from murine M2 cells alone do not react with human cytokines or chemokines.