Figure 6
Chemerin inhibits the production of GM-CSF and IL-6 and NF-κB activation in tumor cells and tumor-associated ECs, respectively, which is mediated by its receptors. (a) Left, the gating strategy and the lineage marker used for isolation of individual cell populations from Hepa1-6 tumor tissue. Epithelial tumor cells (EpCAM+), leukocytes (CD45+), ECs (CD31+PGDFR1α+) and fibroblasts (Fb) (CD31-PGDFR1α+). Right, quantitative reverse transcriptase (qRT)–PCR analysis of gene expression of GM-CSF and IL-6 in individual cell populations sorted from Hepa1-6 tumor tissues. To quantitate the absolute gene expression of GM-CSF or IL-6 by individual cell populations in tumors, the values of 2ΔC(t) of each gene multiply the percentage of individual cell populations in tumors. n=3. (b, c) qRT–PCR analysis of gene expression of GM-CSF and IL-6 (b) and representative western blots showing phosphorylated p65 (p-p65) (c) in tumor cells and tumor-associated ECs, respectively. Tumor cells and tumor-associated ECs were isolated from Hepa1-6 tumors of WT and Rarres2−/− mice, as well as from Ctrl-Hepa1-6 tumors and chemerin- Hepa1-6 tumors of WT mice. n=3. (d) Representative flow cytometry data of the expression of CMKLR1 and CCRL2 in individual cell populations sorted from Hepa1-6 tumor tissues in WT mice. (e) GM-CSF concentrations (upper) and p-p65 levels (lower) in Ctri-Hepa1-6 cells and chemerin-overexpressing Hepa1-6 cells, which were transfected with scramble small interfering RNA (siRNA) or Cmklr1 siRNA or Ccrl2 siRNA. (f) IL-6 concentrations (upper) and p-p65 levels (lower) in bEND.3 EC cells. bEND.3 cells were stimulated with or without supernatants from Ctrl-Hepa1-6 cells or chemerin-overexpressing Hepa1-6 cells, which were transfected with scramble siRNA or Cmklr1 siRNA or Ccrl2 siRNA. Columns and error bars represent of three replicate wells from a representative of three independent experiments. ***P<0.001.
