Fig. 3

PAI-1, rather than IL-6 induces PLOD2 activation and promotion of collagen alignment in CAAs. a Heatmap representing the intensities of each spot of cytokines in the medium. MDA-MB-231 cells were co-cultured with adipocytes for 3 days. Then medium from co-cultured group, cancer alone and adipocytes alone group were collected, and membrane based assays were used to detect cytokines in the medium. b, c) mRNA expression of cytokines in MDA-MB-231 cultured alone or with adipocytes (MDA-MB-231-CO), and adipocytes cultured alone or with MDA-MB-231for 72 h by qPCR. Data was normalized to 1 for monocultures using GAPDH as an internal control. d Expression of PLOD2 fed with indicated PAI-1and IL-6 recombinant proteins. e Immunoblot assay of adipocytes cultured alone or cocultured with MDA-MB-231 cells treated with 5 μg/μl IL-6 neutralizing antibody or tiplaxtinin (20 μM) for PLOD2 expression. f Immunofluorescence staining of type I collagen antibody (green) and DAPI (blue) of adipocytes (control), CAAs (cocultured) or CAAs treated with tiplaxtinin (20 μM) for 72 h, or adipocytes treated with PAI-1 recombinant protein (100 ng/ml, 200 ng/ml) for 72 h. g PAI-1 concentrations in the medium conditioned by 72 h of culturing of MDA-MB-231or SKBR-3, adipocytes, or adipocytes-cancer cells cocultured were analyzed using Elisa. h Western blot analysis of PLOD2 in cancer cells (MDA-MB-231 and SKBR-3) of the conditioned medium (CM) – treated adipocytes. i PAI-1 receptors of adipocytes were analyzed using qPCR assay. j Analysis of protein expression of LRP-1 was done using western blots with extracts obtained from adipocytes cocultivated in the presence or absence of tumor cells, which were treated with tiplaxtinin for 72 h. k Analysis of protein expression of PLOD2 from adipocytes cocultivated in the presence or absence of tumor cells was done using western blots, which were treated with RAP (10 μg/ml) for 72 h