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Fig. 5 | Breast Cancer Research

Fig. 5

From: Exosomal Wnt7a from a low metastatic subclone promotes lung metastasis of a highly metastatic subclone in the murine 4t1 breast cancer

Fig. 5

Wnt7a is a possible candidate protein that plays a role in the enhanced HM.4T1 cell lung metastasis by LM.4T1 cells. A A heat map was generated using normalized RNA-seq data (ID: GSE63180) (Wagenblast et al., 2015) that we obtained from the NCBI GEO (Gene Expression Omnibus, National Center for Biotechnology Information, US National Library of Medicine) database. Gene enrichment analysis in 23 clones from 4T1 cells separated in two subgroups: One displays epithelial features and another displays mesenchymal features. The image shows top five different expression genes between two different groups (ranked by adjusted p values). B Wnt7a mRNA expression in LM.4T1 and HM.4T1 cells was examined by RT-PCR. The results are presented as the mean ± SEM. Statistical significance was analyzed by unpaired Student’s t test. **p < 0.01, n = 4. C Wnt7a protein levels in LM.4T1 and HM.4T1 cell lysates were evaluated by western blotting. The results are presented as the mean ± SEM. Statistical significance was analyzed by unpaired Student’s t test. **p < 0.01, n = 4. D Wnt7a protein levels in LM.4T1 and HM.4T1 cell-derived exosomes were evaluated by western blotting. E Parental 4T1, LM.4T1, and HM.4T1 cells were cultured for 4 days with or without recombinant Wnt7a protein (100 ng/ml). The diameter of each spheroid was measured by the Image J software. Scale bar indicates 100 μm. The results are presented as the mean ± SEM. Statistical significance was analyzed by two-way ANOVA with multiple comparisons. *p < 0.05, **p < 0.01, ****p < 0.0001, n = 6. F The presence of Wnt7a in exosomes derived from wild type or Wnt7a (−) LM.4T1 was examined by western blotting. G Fifty thousand HM.4T1 cells were mixed with the same number of control LM.4T1 cells or Wnt7a (−) LM.4T1 cells and implanted into the 3rd mammary pad of BALB/c nude mice. The size of each tumor was measured, and the tumor volume was calculated. After 28 days of monitoring the tumor. n = 7. H Four weeks after the injection, tumor tissues were excised and weighted. The results are presented as the mean ± SEM. Statistical significance was analyzed by Student’s t test. n = 7. I The number of metastatic tumor nodules was counted 4 weeks after the injection. The results are presented as the mean ± SEM. Statistical significance was analyzed by unpaired Student’s t test. *p < 0.05, n = 7

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