Merkel Cell Polyomavirus–Positive Merkel Cell Carcinoma Requires Viral Small T- Antigen for Cell Proliferation Masahiro Shuda, Yuan Chang, Patrick S.

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Merkel Cell Polyomavirus–Positive Merkel Cell Carcinoma Requires Viral Small T- Antigen for Cell Proliferation Masahiro Shuda, Yuan Chang, Patrick S. Moore Journal of Investigative Dermatology Volume 134, Issue 5, Pages 1479-1481 (May 2014) DOI: 10.1038/jid.2013.483 Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Detection of Merkel cell polyomavirus (MCV) small T (sT) antigen expression by multiple MCV T-antigen antibodies. MCV-positive Merkel cell carcinoma (MCC) cells (MKL-1, MKL-2, MS-1, and WaGa) and MCV-negative UISO cells transfected with MCV genomic T-antigen gene or empty vector as positive and negative controls, were immunoblotted with multiple MCV T-antigen antibodies. α-Tubulin was used as a loading control. Both large T (LT, closed arrows) and small T (sT, open arrows) were detected by CM8E6 and 2T2, sT by CM5E1, and LT by CM2B4. Asterisks indicate non-specific bands. Peptide sequences used for mAb production and shRNA targeting sites are shown in the bottom diagram of T-antigen transcripts with a DnaJ (green box), an Rb-binding (black box) as well as large T stabilization (LSD, yellow box (Kwun et al., 2013)) domains. Journal of Investigative Dermatology 2014 134, 1479-1481DOI: (10.1038/jid.2013.483) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Merkel cell polyomavirus (MCV) small T (sT) antigen knockdown inhibits MCV-positive Merkel cell carcinoma (MCC) cell proliferation. (a) MCV-positive MCC cell lines, MKL-1 and MKL-2, were transduced with pLKO.1-based lentiviral shRNAs targeting both LT and sT (panT.MS) or sT alone (sT1.MS, sT1.RH, and sT2.RH) as described (Houben et al., 2010). Both LT (closed arrows) and sT (open arrows) proteins are detected by 2T2. (b) Expression of LT and sT was quantitated by LI-COR IR immunoblotting system using α-Tubulin for normalization. Relative sT expression to sh ctrl is shown. (c) Small hairpin RNA (shRNA)-transduced MCV-positive (MKL-1 and MKL-2) cells and MCV-negative (UISO) cells were subjected to Wst-1 cell proliferation assay. Error bars indicate SD. Journal of Investigative Dermatology 2014 134, 1479-1481DOI: (10.1038/jid.2013.483) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions