Fig 2: Knockdown of CCL28 expression in endometrial stromal cells by lentiviral transduction. (A) endometrial stromal cells were identified via immunocytochemistry, which was used to analyze CK19 and vimentin expression (×200, 50 µm). (B) CD10 and CD90 were detected using flow cytometry to identify the percentage of EM stromal cells. shCCL28-1, -2 and -3 were constructed to transduce endometrial stromal cells, CCL28 (C) mRNA and (D) protein expression levels were detected to determine the knockdown efficiency of the constructs. **P<0.01 and ***P<0.001 vs. shNC. Control, cells cultured with medium; shNC, cells infected with negative control lentivirus; shCCL28-1, -2 and -3, cells transduced with shCCL28 lentivirus-1, -2 and -3; CCL28, C-C motif chemokine ligand 28; sh, short hairpin RNA; CK19, cytokeratin-19.

Fig 3: Stable transfection of CCL28 cDNA into MDA-MB-231HM cells. (A) Expression of CCL28 mRNA was detected by RT-PCR in MDA-MB-231HM cells that were transfected with control (231HM/vector) or CCL28 cDNA (231HM/CCL28). Expression of CCL28 protein was detected by western blot analysis in 231HM/vector and 231HM/CCL28 cell lines (B) and immunohistochemistry analysis in xenografts of 231HM/vector and 231HM/CCL28 tumor bearing mice (C).

Fig 4: CCL28 enhances breast cancer cell migration, invasion and tumor metastasis. (A) Transwell assay of MDA-MB-231HM/vector (231HM/vector) and MDA-MB-231HM/CCL28 (231HM/CCL28) cells plated on the upper cell culture inserts, with culture medium alone plated in the lower chambers. Quantification of the Transwell assay (right). Cells were counted in triplicate wells and in three identical experiments. **p<0.01. Columns indicate the mean of three independent experiments; bars SD; HP, high-power objective. (B) Cell migration of 231HM/CCL28 cells in a wound-healing assay. Cell migration was assayed at a magnification of ×10. Quantitative analysis of the wound-healing assay (right). Data from three independent experiments were used in this analysis. Values shown are means ± standard deviation (SD). *p<0.05 compared with the untreated group. (C) H&E staining of pulmonary and lymph node metastatic tumors (left). Quantification of the average metastasis of each lung or lymph node of the control xenografts and CCL28-modified xenografts (right). *p<0.05. H&E, hematoxylin and eosin staining.

Fig 5: The MAPK pathway is involved in CCL28-mediated tumor metastasis. (A) Western blot analysis of MMP2, MMP9, VEGF and ß-catenin expression in MDA-MB-231HM/vector (231HM/vector) and MDA-MB-231HM/CCL28 (231HM/CCL28) cells. (B) ß-catenin protein expression was detected by immunohistochemical analysis in xenografts of 231HM/vector and 231HM/CCL28 tumor-bearing mice. (C) Association of CCL28 with the MAPK signaling pathway. Overexpression of CCL28 increased the level of phosphorylated ERK1/2, MEK1/2, and JNK1/2 as determined by western blot analysis. (D) Immunohistochemical examination of pERK protein expression in xenografts of 231HM/vector and 231HM/CCL28 tumor-bearing mice. MAPK, mitogen-activated protein kinase; MMP, matrix metalloproteinase; VEGF, vascular endothelial growth factor.