Loss of Novel mda-7 Splice Variant (mda-7s) Expression is Associated with Metastatic Melanoma1 Matthew Allen, Barbara Pratscher, Florian Roka, Clemens.

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Loss of Novel mda-7 Splice Variant (mda-7s) Expression is Associated with Metastatic Melanoma1 Matthew Allen, Barbara Pratscher, Florian Roka, Clemens Krepler, Volker Wacheck, Christian Schöfer, Hubert Pehamberger, Markus Müller, Trevor Lucas Journal of Investigative Dermatology Volume 123, Issue 3, Pages 583-588 (September 2004) DOI: 10.1111/j.0022-202X.2004.23321.x Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Differential splicing of mda-7 RNA. The mda-7 splice variant (mda-7s) was amplified and cloned from normal human melanocytes. (a) mda-7s is spliced (↓) aberrantly between exons (E) 2 and 4, and exons 4 and 6 shown in the sequence traces (b) generating a reading frame between exons 2 and 7. Homologies between the 206 residue wild-type MDA-7 and the 63 amino acid MDA-7S are restricted to 14 residues (underlined) derived from exon 2. Journal of Investigative Dermatology 2004 123, 583-588DOI: (10.1111/j.0022-202X.2004.23321.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Functional interaction between MDA-7 and MDA-7S. HEK293 cells were transiently cotransfected with HA-tagged MDA-7S (pMHmda-7sHA), wild-type mda-7 as a C-terminal GFP fusion protein (pmda-7CTGFP), or the empty vectors pCTGFP and pMH-HA. Cell lysates were precipitated either on anti-HA agarose (a) or with anti-GFP antibodies on protein G (b) and supernatants were western blotted and probed with monoclonal antibodies against GFP (a) or HA (b). MDA-7S-HA coprecipitated the 62 kDa MDA-7CTGFP protein (a) and MDA-7CTGFP, the 12 kDa MDA-7S-HA polypeptide (b), demonstrating functional interaction between the proteins. (c) HEK293 cells were transfected with pmda-7CTGFP and either pMHmda-7sHA or pMH-HA and the levels of secreted MDA-7CTGFP were assessed in the medium by western blotting. Coexpression of MDA-7CTGFP and MDA-7S-HA significantly reduced secretion of MDA-7CTGFP. Journal of Investigative Dermatology 2004 123, 583-588DOI: (10.1111/j.0022-202X.2004.23321.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Expression of mda-7s during melanoma development. Expression of mda-7, mda-7s, and β-tubulin mRNA expression was analyzed by PCR in normal human melanocytes (a, lanes 1–4), spontaneously transformed melanocytes (a, lanes 5–7), nevi (b, lanes 1–3), lymph node metastasis (c, lanes 1–3), subcutaneous metastasis (c, lanes 4–6) and the melanoma cell lines A375, 607B and MelJUSO (d, lanes 1–3). In real-time PCR amplification of mda-7s (e), representative graphs show expression levels in normal melanocytes (I) decreasing to undetectable levels in metastatic melanoma samples within 40 cycles of amplification (II). Normalized reporter signal represents fluorescence changes adjusted to baseline before amplification and (Ct) the threshold cycle of amplified products. Journal of Investigative Dermatology 2004 123, 583-588DOI: (10.1111/j.0022-202X.2004.23321.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions