ERK and MCT-1 in DLBCL cell lines and primary cells. (A) SUDHL6, SUDHL10, OCI-LY19, and OCI-LY3 cells were treated with 200nM AZD6244 for the indicated periods of time. Cell lysates were subjected to Western blotting using pERK and ERK antibodies. Actin was used as an internal control. (B) SUDHL4, SUDHL6, and OCI-LY19 cells were treated with indicated concentration of AZD6244 for 18 hours. pERK, ERK, and MCT-1 protein levels were measured by Western blotting using the respective specific antibodies. (C) Western blot showing down-regulation of pERK and MCT-1 in primary DLBCL cells afterAZD6244 exposure for 6 and 16 hours. Peripheral blood monocytes (PBMC) were obtained from 3 DLBCL patients (each with relapsed/refractory transformed DLBCL with leukemia involvement). PBMCs were incubated with indicated concentrations of AZD6244 for 6 or 16 hours. This patient/figure is representative of 3 primary DLBCL subjects. Cell lysates were subjected to Western blotting using specific antibodies for pERK, ERK or MCT-1. (D) Over-expression of constitutively active (CA) MEK2 increased ERK and MCT-1 protein levels. Raji cells were transfected with wild-type (WT) MEK2, CA MEK2 construct, or the vector control (V). After 24 hour transfection, cells were incubated with 200nM of AZD6244 for 24 hours. The cell lysates were subjected to Western blot analysis. (E) OCI-LY3 cells were transduced with scrambled (sc) shRNA or ERK2 shRNA by spin infection using GIPZ lentivirus system. After puromycin selection cells were subjected to Western blotting to check the protein level. (F) OCI-LY3 cells after positive selection were treated with 200nM or 300nM AZD6244 for 48 hours, which was followed by annexin V/PI staining and analyzed by flow cytometry.