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Fig. 7. MCP-1 and IL-8 overexpression following Cdc42 silencing. (A) Analysis of serum-free medium conditioned for 16 hours between days 2 and 3 after transfection by cytokine proteoarray. HSFs were transfected with 20 nM siRNA targeting RhoA (siRhoA), Rac1 (siRac1) or Cdc42 (siCdc42), or with 20 nM irrelevant siRNA (siScr). White arrows indicate the positions of the IL-8, IL-6 and MCP-1 signals, respectively, from top to bottom (B) Representative semiquantitative RT-PCR analysis of MCP-1 and IL-8 mRNA level and of 28S rRNA in total RNA extracted from HSF 72 hours after transfection with calcium phosphate alone (CaP), an irrelevant siRNA (siScr) or an siRNA targeting RhoA (siRhoA), Rac1 (siRac1) or Cdc42 (siCdc42). The efficiency of the 28S RT-PCR is controlled using a known copy number of synthetic RNA co-transcribed and co-amplified with the same primers to generate a product of larger size (*). (C) ELISA measurements of MCP-1 and IL-8 in serum-free medium conditioned by HSFs for 16 hours between days 2 and 3 after transfection.

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