Figure 6

Knockdown of Fascin in LMP1-transfected Jurkat cells diminishes invasion through extracellular matrix. (A) Jurkat cells were transfected with pMACS-LNGFR, wt-LMP1 (pSV-LMP1) and shFascin5, shFascin4 or shNonsense (shNon) and subjected to magnetic separation. Immunoblots of Fascin, LMP1 and ACTB are shown. Numbers indicate the relative expression of Fascin normalized on ACTB as determined by densitometry (at least 4 experiments). Values obtained from cells transfected with LMP1 and a control shRNA (shNon) were set as 1. indicates cross reactions of the LMP1-antibody. (B) Invasion assays were performed with serum-starved Jurkat cells (1% FCS) after magnetic separation 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. Mock-transfected cells were set as 100%. Mean values and error bars of three independent experiments each performed in quadruplicates are shown. Values were compared using a paired t-test. **indicates P < 0.01. *indicates P < 0.05. n.s., not significant.