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. 2013 Sep 19;9(9):e1003789. doi: 10.1371/journal.pgen.1003789

Figure 3. shRNA silencing of CCL2, CCL7 or CCL8 in tumor-supportive fibroblasts reduces their ability to promote tumorigenicity.

Figure 3

(A) Reduction of CCL2 protein in HFFF2 fibroblasts by expressing shRNAs targeting CCL2 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 CCL7. (C) Reduction in CCL8 RNA levels in HFFF2 fibroblasts expressing shRNAs targeting CCL8 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 CCL2. 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 CCL7 (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 CCL8 (p<0.01). (G) Tumorigenicity of MDAMB231 cells alone or co-injected with HFFF2 cells expressing either no shRNA, control shRNA or shRNAs targeting CCL2. 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 CCL7 (p<0.01). (I) Effects of shRNAs targeting CCL8 (p<0.01). The lack of effects of the CCL-targeting shRNAs on fibroblast proliferation are shown in Figure S4.