shRNA silencing of <i>CCL2</i>, <i>CCL7</i> or <i>CCL8</i> in tumor-supportive fibroblasts reduces their ability to promote tumorigenicity.

<p>(A) Reduction of CCL2 protein in HFFF2 fibroblasts by expressing shRNAs targeting <i>CCL2</i> as indicated compared to non-targeting (N.T.) control determined by immunoblotting. Beta-actin was used as a loading control. (B) Reduction of CCL7 protein levels in HFFF2 fibroblasts expressing shRNAs targeting <i>CCL7</i>. (C) Reduction in <i>CCL8</i> RNA levels in HFFF2 fibroblasts expressing shRNAs targeting <i>CCL8</i> compared to HFFF2 fibroblasts expressing non-targeting (N.T.) control. Asterisk indicates a significant difference (p<0.01) between the experimental (shCCL8) and control (shN.T.) group. (D) Tumorigenicity of Cal51 cells alone or co-injected with HFFF2 cells expressing either no shRNA, control shRNA or shRNAs targeting <i>CCL2</i>. Tumor-take rate for the total injections for each group is indicated. Asterisks indicate significant differences (p<0.01) in tumor volumes for shCCL2-1 and shCCL2-2 compared to control. Hashtag indicates a significant difference (p<0.05) between cell line only (Cal51) and Cal51 co-injected with HFFF2 fibroblasts. Error bars represent SEM. (E) Effects of shRNAs targeting <i>CCL7</i> (p<0.01). There was no significant difference in the tumor volumes between the two knockdown groups (p = 0.3, 0.3, 0.2 and 0.5 at weeks 3, 4, 5 and 6 post injections respectively). (F) Effects of shRNAs targeting <i>CCL8</i> (p<0.01). (G) Tumorigenicity of MDAMB231 cells alone or co-injected with HFFF2 cells expressing either no shRNA, control shRNA or shRNAs targeting <i>CCL2</i>. Tumor-take rate for the total injections for each group is indicated. Asterisks indicate significant differences in tumor volumes for shCCL2-1 and shCCL2-2 compared to control (p<0.01). Hashtag indicates a significant difference between cell line only (MDAMB231) and MDAMB231 coinjected with HFFF2 fibroblasts (p<0.05 and n = 10 per group). Errors represent SEM. (H) Effects of shRNAs targeting <i>CCL7</i> (p<0.01). (I) Effects of shRNAs targeting <i>CCL8</i> (p<0.01). The lack of effects of the CCL-targeting shRNAs on fibroblast proliferation are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003789#pgen.1003789.s004" target="_blank">Figure S4</a>.