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. 2011 Apr 25;108(19):7820–7825. doi: 10.1073/pnas.1103441108

Fig. 1.

Fig. 1.

Silencing Sca-1 expression decreases colony formation and tumorigenic potential, and increases GDF10 expression. 34T cells were transduced with a lentivirus expressing either GFP shRNA as a control or a Sca-1 shRNA (Fig. S2). D8 cells expressing Sca-1 shRNA cells exhibited a 90% reduction in Sca-1 expression compared with control 34T cells, as assessed by FACS analysis (A) or Western blot analysis (B). (C) Down-regulation of Sca-1 expression reduces spheroid cluster morphology. 34T cells grew as spheroid clusters, whereas D8 cells grew as a flattened monolayer with contact-inhibited growth. (D) Silencing Sca-1 expression reduces colony formation. Colony-formation assay in D8 cells vs. 34T cells showed that growth was reduced by 70% in D8 cells vs. 34T cells. (E) Down-regulation of Sca-1 expression markedly reduces tumorigenicity. 34T cells and D8 cells were implanted s.c. at an inoculum of 100,000 cells into opposite flanks of eight syngeneic C57BL/6 mice. Tumor formation was monitored over 25 d, and tumor volume was calculated. Isografts from D8 cells exhibited little growth compared with isografts from 34T cells (P < 0.001, two-tailed Student t test). (F) Conditioned medium (CM) from 34T cells enhanced colony formation of D8 cells, whereas conditioned medium from D8 cells markedly reduced colony formation of 34T cells. The bar graph shows quantification of colony growth; the number in each bar corresponds to the plate number in the left panel. (G) Real-time qPCR analysis of TGF-β ligands. The fold increase in expression of each ligand in D8 cells relative to 34T cells is shown. D8 cells expressed >100-fold higher levels of GDF10 mRNA compared with 34T cells. (H) 34T/GFP shRNA cells stably expressing GDF10 (34T/GDF10) exhibit a >100-fold increase in GDF10 mRNA expression compared with control cells (34T). (I) D8 cells transfected with a GDF10 shRNA (D8/GDF10sh) show 90% reduced GDF10 mRNA expression compared with cells expressing a control shRNA (D8). (J) Modulation of GDF10 expression results in changes in colony formation. Expression of GDF10 in 34T cells (34T/GDF10) reduced colony formation by 80%, whereas silencing GDF10 expression in D8 cells (D8/GDF10sh) increased colony formation. (K) GDF10 modulates tumorigenicity. Cells were implanted into eight syngeneic C57BL/6 mice, and tumor growth was assessed as in E. Tumor volume was reduced in isografts of 34T/GDF10 cells compared with tumors from 34T cells (P < 0.001, two-tailed Student t test). In contrast, D8/GDF10sh cells showed increased tumor volume compared with D8 cells (P < 0.001, two-tailed Student t test).