Figure 4.

p90 ribosomal S6 kinase (RSK) inhibition suppresses a promotile transcriptional program. A: Heat map demonstrating down-regulation of 200 mRNAs involved in cell motility and/or cell adhesion after RSK inhibition. Yellow bars indicate the basal mRNA expression level. Blue bars indicate mRNA down-regulated ≥1.33 fold (P < 0.005) compared with dimethyl sulfoxide (DMSO)–treated cells. B: Venn diagram classifying 200 mRNAs that were down-regulated after RSK inhibition and functionally annotated with biological process terms related to cell motility and/or cell adhesion. C: Results from mRNA array demonstrating down-regulated expression of matrix metalloproteinase 2 (MMP-2) and five other related transcripts after 24 or 48 hours of treatment with BI-D1870. Data were normalized by setting DMSO-treated cells to 100%, and microarray was confirmed using quantitative RT-PCR (RT-qPCR). D: Human MMP antibody array comparing four superficial spreading melanoma (SSM) and four nodular melanoma (NM) metastatic samples. Boxes indicated correspond to tissue inhibitor of metalloproteinases 1 (TIMP-1) and MMP-8 as indicated in the key. E: Quantitative spot density analysis of the array shown in D. The intensity of each spot was normalized to the positive control, and duplicate values were evaluated as a mean. F: Validation of the array using immunohistochemical analysis. NM metastatic tissue showed higher expression of MMP-8 relative to SSM metastatic tissue. Samples were scored based on intensity (0 to 2) (mean of nine metastatic SSM samples and 10 metastatic NM samples). Data are given as means ± SEM. ^∗P < 0.05. Original magnification: ×20 (F); ×40 (insets, F).