(A) mRNA from transduced MDA-MB435 cells was used to probe a microarray representative of the human genome

Results are shown as a dot plot of individual genes, comparing the expression levels found in EV– and SEMA3B-transduced cells. A selected panel of 622 genes, including cytokines and factors mediating inflammatory response (identified by keywords as described in Materials and methods), is highlighted in red. is the only gene in the panel significantly regulated more than twofold. ***, P < 0.001) (B) Real-time quantitative PCR mRNA expression analysis of SEMA3B- and EV-transduced tumor cells using Taqman probes for IL-8 and for the housekeeping gene (internal reference). Reported values indicate fold induction of IL-8 expression in SEMA3B-expressing cells relative to the respective EV control for each cell line, and values correspond to the mean ± SD of three individual experiments. *, P < 0.05. (C) The CM from MDA-MB435 EV control and SEMA3B-expressing cells was collected and concentrated 300 times. 10 μl of this preparation was analyzed by Western blotting with anti–IL-8 antibodies. The indicated amounts of purified IL-8 were loaded for internal reference. The concentration of IL-8 in the original CM of MDA-3B cells was thus estimated to be in the range of 5–7 ng/ml. (D) Western blot analysis of A549 cells expressing SEMA3B under a drug-inducible promoter; control cells (EV) expressed the doxycycline-dependent transactivator rTTA alone. Upon doxycycline treatment, SEMA3B expression in tumor cells (revealed by anti-Myc tag antibodies) and IL-8 levels in the CM (revealed with MAB208) were increased; purified IL8 was loaded as an internal reference. (E) The CM of SEMA3B tumor cells and EV controls were assayed in monocyte migration experiments, similar to those shown in . Where indicated, IL-8 neutralizing antibodies (MAB208) or an unrelated control antibody (i.e., anti-CD19; C Ab), or 100 ng/ml of purified IL-8 were added in the lower well. The number of migrated monocytes was scored as in , and the graph shows the mean of three independent experiments. IL-8 is responsible for the increased monocyte-attracting activity released by SEMA3B-expressing tumor cells. **, P < 0.01.Copyright information:Taken from "The tumor suppressor semaphorin 3B triggers a prometastatic program mediated by interleukin 8 and the tumor microenvironment"The Journal of Experimental Medicine 2008;205(5):1155-1171.Published online 12 May 2008PMCID:PMC2373847.