Wnt5a/ROR1 induces production of proinflammatory factors that lead to phosphorylation of STAT3 in CLL cells. (A) Immunoblot analysis of anti-pSTAT3(Y705) or anti-STAT3 in lysates of CLL cells treated with Wnt5a (200 ng/ml) for 0-6 hours or 24 hours in the presence of control IgG or cirmtuzumab. Bar figures represent ratios of density (pSTAT3/Total STAT3). (B) Wnt5a/ROR1 induced production of cytokines in CLL cells. The medium of CLL cells cultured with or without Wnt5a for 16 hours was analyzed by the human cytokine array. (C) Cirmtuzumab or neutralizing anti-Wnt5a antibodies blocked Wnt5a-induced cytokine production in CLL cells. The medium of CLL cells cultured with or without Wnt5a, in the presence of control IgG, cirmtuzumab, or anti-Wnt5a was analyzed by the human cytokine array. (D) Immunoblot analysis of anti-pSTAT3 (Y705) or anti-STAT3 in lysates of CLL cells treated with control medium, conditioned medium, or IL-6. Tocilizumab or control IgG was added to the CLL cultures 2 hours before treatment. The numbers on the top indicate the relative density of pSTAT3/Total STAT3. The density of the sample treated by conditioned medium only (the 3rd lane) was considered as 1.0. (E) Reverse transcript PCR analysis of IL-6 and β-actin in RNA isolated from purified CLL cells treated with Wnt5a (200 ng/mL) in 0 to 5 hours. (F) ELISA of IL-6 in medium of isolated CLL cells cultured with Wnt5a for 0 to 8 hours in the presence of cirmtuzumab or control IgG (n = 5).