Table 1.
Primer's sequences used in this study and their annealing optimal temperature and time.
Figure 1.
Number of MDSCs in mammary tumor-bearing dogs, their IL-28 expression and changes in canine mammary tumor cells gene/protein expression due to their co-culture with MDSCs.
(A). The number of circulating MDSCs (%) within white blood cells in healthy donors, and dogs with stage I, II, III and IV mammary cancer. Results that differed significantly compared to control are marked by ‘a’ (P<0.01) or ‘b’ (P<0.001). (B). Volcano plot of gene expression in canine mammary tumor cells co-cultured with MDSCs compared to monocultured cells. Genes which expression differed significantly (P<0.05, FC>2.0) are marked by blue dots. Il-28ra is marked as a red dot. The plot was generated using BRB software. (C). Changes in expression of selected genes in canine mammary tumor cells due to co-culture with MDSCs visualized on agarose gel using UV light (ctrl, control cells grown as mono-culture; +MDSC, cells grown as co-culture with MDSCs). (D). Relative Il-28ra gene expression in canine mammary tumor control cells transfected with non-coding siRNA (ctrl), cells co-cultured with MDSCs (+MDSC), transfected with Il-28ra-specific siRNA (siRNA) and treated with IL-28 (IL28). Results that differ significantly compared to control are marked as ‘a’ (P<0.001), ‘b’ (P<0.05) or ‘c’ (P<0.01). (E). Relative gene expression of Il-28 in MDSCs isolated from blood of healthy dogs, and dogs with stage I/II or III/IV mammary cancer. (F). Expression of selected target and downstream signaling proteins in control cells mock-transfected (ctrl), cells with knockdown of IL-28RA expression (siRNA) and cells treated with IL-28 (IL28).
Figure 2.
Vessel formation (3D) by HUVECs.
Representative pictures showing 3D vessel formation by HUVECs due to co-culture with P114 canine mammary control cells (mock-transfected), cells treated with Il-28ra-specific siRNA, cells treated with IL-28 (before the experiment), cells treated with IL-28 during the experiment and positive and negative control cells, as well as only IL-28 treatment.
Figure 3.
Changes in cytokeratin and vimentin expression.
Representative pictures showing changes in expression of cytokeratin (upper panel) and vimentin (bottom panel) in P114 canine mammary control cells (mock-transfected), cells treated with Il-28ra-specific siRNA and cells treated with IL-28. Values that differed significantly are marked as * (P<0.05) or *** (P<0.001).
Figure 4.
Growth characteristics on Matrigel matrix.
Phase contrast micrographs of CMT-U27, CMT-U309, and P114 cells grown on the Matrigel matrix under control conditions or treated with il-28ra siRNA either IL-28. Control and siRNA-treated neoplastic cells formed colonies, whereas those treated with IL-28 changed their shape for spindle-like, invaded the Matrigel matrix and formed branches.
Figure 5.
Migration and invasion in vitro assay.
Representative pictures showing invaded (upper panel) and migrated (bottom panel) CMT-U27 canine mammary tumor cells: control (mock-transfected) (NC), treated with IL-28 (IL28) or treated with Il-28ra-specific siRNA (siRNA). Values that differed significantly are marked as * (P<0.05), ** (P<0.01) or *** (P<0.001).