Inhibitory effect of LM-234ep conditioned medium on the proliferation of LM-234mf cells. LM-234mf and LM-234ep cells were seeded into 6-well plates at a density of 1 × 104 cells/well and allowed to attach overnight. Thereafter, cells were incubated in the presence of LM-234ep CM (black circles), LM-234mf CM (black triangles), or culture medium (black squares) supplemented with 10% FBS. Cell numbers were counted at different time points, and expressed as mean ± SE. * P = 0.05, ** P = 0.0018 (Student's t Test). Assays were performed in triplicate, (A). LM-234mf cells were incubated with culture medium + 10% FBS (Control) or LM-234ep CM for 72 h. Cells were processed for DNA content, and cell cycle progression was analyzed by flow cytometry (B). LM-234mf cells were treated with culture medium (Control) or LM-234ep CM for the times indicated (top). Cells were collected, lysed, and analyzed by immunoblotting using antibodies specific for p-ERK, c-Jun, JunB, cyclins E, A, and D, and Cdk2. β-actin was used as a loading control (C). Immunofluorescence for c-Jun, Cyclin A and Cyclin D in LM-234mf cells. The nuclear localization of the proteins is shown in the control cells (white arrows). Magnification bar, 40 μm (D). AP-1 luciferase activity in LM-234mf cells co-transfected with AP-1-Luc and pRL-tk-luc and treated with LM-234ep CM or culture medium (Control). ** P = 0.005 (Student's t Test). The results shown are the mean ± S.D. of three experiments (E).