Figure 6.

The effects of CCM111 on NO production and the protein expression of iNOS, COX-2, and inflammatory cytokines in murine RAW264.7 macrophages. The cells were treated with LPS (100 ng/ml) alone or in combination with different concentrations of CCM111. After incubation for 24 hours, the culture media was collected for nitrate assay analysis as shown in (a), and the protein lysates were analyzed by immunoblotting for iNOS, COX2 and β-actin as shown in (b) The full length blots images are presented in Supplementary Fig. 5a. Results in (a) and (b) were obtained from three independent replicates. The cells were treated with LPS (100 ng/ml) alone or in combination with LPS and CCM111 (160 μg/ml) for 24 h. The cell lysates were analyzed by the RayBio C-series mouse cytokine antibody array C1. (c) The left panel shows an image of the signal spots on the membrane for each cytokine. Each spot represents one cytokine, and each cytokine is in duplicate. Spots that show significant change are marked. The right panel shows the identities of all spots on the array with coordinates. The full length blots images are presented in Supplementary Fig. 5b. The left image of spots was scanned and measured by NIH Image-J software. (d) The relative expression level of each protein was calculated from densitometry data from (c) and normalized to the LPS treatment alone array. The blue dotted line represents a 0.8-fold difference, and the red dotted line represents a 1.2-fold difference. Error bars indicate S.D. (n = 2).