Fig. 7. TPL2 activity is required for proliferation and survival of EBV-transformed human B cells.

a IKK2 and TPL2 inhibition causes downregulation of the JNK pathway in lymphoblastoid cell lines (LCLs). Quantitative immunoblot analysis of LCL721 cells kept in the presence of DMSO, 10 µM of ACHP (left panels) or TPL2-IH (right panels) for 3 h. The indicated antibodies were used. Paired signals standardized to total protein levels of three biological replicates are shown. Chemiluminescence values (y-axes) are relative and do not allow for direct comparison between graphs. b TPL2 inhibition retards proliferation of LCLs. LCL721 and LCL 1C3 cells were incubated in the absence or presence of DMSO or 10 µM TC-S7006 (TPL2-IH). Proliferation was determined as MTT conversion. Data are mean values ± SD of three biological replicates. c TPL2 inhibition causes apoptosis in LCLs. Cells were kept in the presence of DMSO or 10 µM TPL2-IH for 24 or 48 h, stained with propidium iodide (PI) and Annexin V-Cy5, and analysed by flow cytometry. FACS plots of one representative experiment after 24 h are shown. Percentages of AnV+/PI− cells indicating apoptotic cells are given in graphs as mean values ± SD of three biological replicates. d Cell death after TPL2 inhibition is specific for EBV-transformed B-cells. LCL721 and EBV-negative BL41 B-cells were grown in the presence or absence of TPL2-IH for 24 h and apoptosis rates were determined. Data are mean values ± SD of three biological replicates. e The activity of caspase 3 is increased in LCLs after TPL2 inhibition. Caspase 3 activity was measured after incubation of cells with DMSO or 10 µM TPL2-IH for 24 h. Staurosporin treatment for 4 h served as positive control and reference. Data represent the x-fold change ± SD of three biological replicates. a Statistical analysis was performed with the ratio paired T-test (two-tailed). b–e Statistical analysis was performed with two-way ANOVA at alpha 0.05. a–e p-values: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, n.s. not significant.