Figure 4

DH82 induces hypoxia in canine MGT cells and affects tumor cell growth factor and multi-drug resistance gene expression under doxorubicin. (A) DH82 infiltration model using CIP alone spheroid. Canine MGT cells were seeded in an ultra-low adhesion plate alone. After spheroid formed, DH82 were added into the well and co-culture for 24 h to infiltrate the spheroid. (B) Immunofluorescence imaging of DH82 infiltrated spheroids. 500 μM pimonidazole was treated for 2 h before fixation. Spheroids were stained with DAPI (blue), FITC dye -conjugated CD11b mouse monoclonal antibody (green) and APC dye -conjugated IgG₁ mouse monoclonal anti-pimonidazole antibody (red) (white bar = 650 μm). (C) Relative hypoxic cell area of spheroids with or without DH82 infiltration. (D) mRNA relative expression of growth factors and multi-drug resistance genes in canine MGT cells co-cultured with or without DH82 under doxorubicin (0, 0.18, and 0.37 μM) for 48 h. The results are presented as the mean ± SD of triplicate samples of three independent experiments. MGT mammary gland tumor, COX2 cyclooxygenase 2, HIF-1α hypoxia-inducible factor 1-alpha, VEGF vascular endothelial growth factor, TGF-β transforming growth factor β, TSG-6 TNF-stimulated gene 6 protein, MRP1 multi-drug resistance-related protein, P-gp P-glycoprotein, DAPI 4,6-diamidino-2-phenylindole, APC allophycocyanin, IgG1 immunoglobulin G1. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, as determined by one-way ANOVA.