Bars in (B) and (C) shows a densitometric scanning of the Western blot signals. activity. Interestingly, the ectopic expression of CCL17/TARC upregulated MCPyV early and late promoter activities in MCC13 cells. Furthermore, recombinant CCL17/TARC activated both the mitogen-activated protein kinase and the NF-B pathways. Finally, immunohistochemical staining on human MCC tissues showed a strong staining of CCL17/TARC and its receptor CCR4 in both LT-positive and -unfavorable MCC. Taken together, CCL17/TARC and CCR4 may be a potential target in MCC therapy providing MCC patients with a better overall survival end result. 0.05, *** 0.001. Expression of CCL17/TARC and CCR4 in MCC cells To confirm the stimulating effect of full-length and truncated LT on CCL17/TARC promoter activity, we first evaluated the mRNA expression of CCL17/TARC by qPCR in MCC13 cells transfected with expression vector for full-length LT or truncated LT variants. Full-length and MKL-1 LT significantly increased CCL17/TARC expression in the MCC13 cell collection (P 0.01), while MKL-2 and MS-1 LT variants, modestly, but significantly increased CCL17/TARC mRNA levels (Physique ?(Figure4A).4A). Western blot analysis with anti-CCL17/TARC antibodies confirmed that CCL17/TARC protein levels were increased in MCC13 cells expressing either full-length or truncated LT compared to MCC13 cells (Physique ?(Physique4B4B and ?and4D).4D). In our screening experiments, we also found a slight upregulation of CCR4 mRNA by exogenous expression of both pcDNA3-FLTA and pcDNA3-MKL-2 plasmids. So, to check at the protein level, we conducted a Western blot. We did not find expression at a significant level, but only a slight upregulation of CCR4 in MCC13 cells with an exogenous expression of MCPyV LT (Physique ?(Physique4C4C and ?and4D4D). Open in a separate window Physique 4 Transient expression of full-length or truncated MCPyV LT increases the transcript and protein levels of CCL17/TARCMCC13 cells were transfected with an empty vector or expression plasmid for MCPyV full-length LT (FLTA), Rabbit Polyclonal to KANK2 or truncated MKL-1, MKL-2, or MS-1 LT. (A) qRT-PCR analysis shows CCL17/TARC mRNA levels normalized with eukaryotic 18S rRNA levels. (B) CCL17/TARC protein Tivozanib (AV-951) levels analyzed by Western blotting. The uttermost right lane represents baseline expression of CCL17/TARC by MCC13 cells. (C) CCR4 protein levels analyzed by Western blotting. A Western blot with ERK2 antibodies was used as a loading control. (D) Representative figures of CCL17/TARC (B) and CCR4 (C) Western blots. The lane most to the left in the lower part contains the protein molecular mass marker (in kDa). 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