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Figure 3 | Virology Journal

Figure 3

From: Modified Vaccinia virus Ankara but not vaccinia virus induces chemokine expression in cells of the monocyte/macrophage lineage

Figure 3

Supernatants from MVA but not VACV WR infected MH-S cells increase chemotaxis of U-937 cells in a CCR1 dependent manner. A) Total RNA from THP-1 cells (T) and U-937 cells (U) were isolated and used for GAPDH, CCR1 and CCR2 transcript variant A specific RT-PCR. B) Total RNA was isolated from cells as indicated and a multiprobe ribonuclease protection assay using hCR-8 (BD Biosciences) as probe was performed as previously described [12]. C) Verification of J 113863 to block CCR1 specific chemotaxis of THP-1 cells. Human CCL3 at concentrations as indicated was added to the lower part of a 96-well Multi-Screen-MIC plate with an 8 μm pore-sized filter (Millipore). 75.000 cells were placed in the upper part and cells migrated into the lower part were counted after 90 min. Data are means ± SD (n = 3). D) Human monocytic THP-1 cells (left panel) and U-937 cells (right panel) were tested for chemotaxis towards control medium (CM) and supernatants from mock, MVA or VACV WR infected MH-S cells (1 MOI, 16 h) as described in C). Where indicated, cells were pre-incubated with 5 nM of the CCR1 antagonist J 113863 for 5 min prior to running the assay. THP-1 cells and U-937 cells were allowed to migrate for 90 min and 30 min, respectively. Data are means ± SD (n = 3 for THP-1 cells; n = 6 for U-937 cells) and are representative of at least two independent experiments, **, P < 0.01; ANOVA with Bonferroni post-hoc test.

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