Figure 5

NF-κB signals are required for maintaining Fascin expression and invasive migration of EBV-transformed LMP1-expressing lymphoblastoid cells (LCL). (A) Viability of LCL-B cells upon treatment with increasing amounts of the IKKβ inhibitor ACHP (1, 2.5, 5, 10, 25 μM) and the JNK-inhibitor SP600125 (10 μM) for 48 h determined by forward-side scatter (FSC/SSC) analysis in flow cytometry. DMSO-treated cells were set as 100%. The means of three independent experiments +/− SE were compared using a paired t-test. *indicates P < 0.05. (B) Quantitative PCR of Fascin transcripts normalized to ACTB in LCL-B upon ACHP-and SP600125-treatment for 48 h. The means of three independent experiments +/− SE were normalized to solvent-treated cells and compared using a paired t-test. **indicates P < 0.01. (C) Detection of Fascin, LMP1, p100 processing (p100/p52) and IκBα by immunoblot after treatment of LCL-B with DMSO, 2.5 and 5 μM ACHP for 48 h. ACTB served as loading control. Samples were loaded on two gels in parallel. (D) LCL-B cells were cultured in presence of ACHP (5 μM) or solvent (DMSO) for 48 h and cells were serum-starved (1% fetal calf serum (FCS)) for 4 h. Invasion assays were performed using trans-wells coated with extracellular matrix for 24 h. Values shown in the upper panel reflect the percentage of invaded cells (measured at OD 560 nm) that are attached to the bottom of the membrane. The lower bar graphs show the percentage of invaded cells that are non-attached and have migrated to the medium (20% FCS) of the lower compartment. Solvent-treated cells (DMSO) were set as 100%. Mean values and error bars of three independent experiments each performed in triplicates are shown. Values were compared using a paired t-test. *indicates P < 0.05. n.s., not significant.