(A) MDA-MB435 (MDA) and A549 cells transduced to express SEMA3B (3B) or a noncoding EV were grown in culture for 4 d in 1% FBS

Cell growth was evaluated daily in separate dishes by staining cells with crystal violet and measuring absorbance. Data shown represent the mean and SD of triplicates.(B and C) SEMA3B-transduced and EV control tumor cells were injected subcutaneously into nude mice. Graphs display tumor volume (measured externally during the experiment; B) and tumor weight (measured after excision at the end of the experiment; C), respectively. Data shown are the mean ± SEM from nine mice per each experimental group. ***, P &lt; 0.0005. (D) The expression of SEMA3B (Myc tagged) was detected in tumors by staining tissue sections with anti-Myc antibodies (green), whereas nuclear counterstaining was done with DAPI (blue). Bar, 100 μm. SEMA3B was similarly detected in tumor lysates by Western blotting (bottom). (E) At the end of the experiment, the lungs of mice carrying tumor xenografts (as described above) were contrasted by airway perfusion with India ink, and superficial metastases were counted under a stereoscopic microscope. Numbers of metastases are given as the mean ± SD from nine mice for each experimental group (left). We further determined the metastatic index of SEMA3B-expressing and control tumors (right) by calculating the ratio between the number of metastatic foci and tumor weight. **, P &lt; 0.01; ***, P &lt; 0.001. (F) A549 cells expressing SEMA3B under control of a doxycycline-inducible promoter were injected subcutaneously in nude mice. Tumor cells expressing the transactivator rTTA alone were used as controls. After 46 d, the mice were killed, and we measured tumor burden, counted lung metastasis, and calculated the metastatic index as described. Data shown are the mean ± SEM. Western blot analysis on protein lysates from tumor samples (top) revealed a minimum level of recombinant SEMA3B expression in the absence of the doxycycline, which was strongly induced by treatment with the drug. *, P &lt; 0.05; **, P &lt; 0.01.<b>Copyright information:</b>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.