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Figure 3 | Breast Cancer Research

Figure 3

From: Tumour-associated tenascin-C isoforms promote breast cancer cell invasion and growth by matrix metalloproteinase-dependent and independent mechanisms

Figure 3

Confirmation of expression of tenascin-C isoforms. (a) Endogenous expression of tenascin-C (TNC) isoforms in untransfected cell lines. Normalised relative expression of TNC isoforms were determined by reverse transcriptase polymerase chain reaction (RT-PCR) using primers and probes to invariant exon 17/18 boundary (Total TNC), the 9/16 (TNC-16) and 14/16 (TNC-14/16) exon boundary for breast cell lines ZR-75-1, MCF-7, T-47D, GI-101, Hs578T, MDA-MB-231, MDA-MB-436, MDA-MB-468 and melanoma cell line SKMel-28. Relative expression was calculated by comparison with standard curves derived from TNC-9-16 and TNC 9–14–16 recombinant clones to correct for differences in PCR efficiency for each TNC probe set normalised using level of housekeeping gene expression to correct for any differences in cellularity. (b) RT-PCR of primary fibroblasts and MCF-7 cells transiently transfected with TNC-S, TNC-L, TNC-16, TNC-14/16 and vector-only control (vector), using primers spanning the FN III alternatively spliced region (8F/18R). This shows appropriately sized bands in each of the cell populations, with no product in vector-only and non-transfected MCF-7 controls, although there was evidence of low level expression of TNC-S in vector only and non-transfected fibroblast controls. (c) Immunohistochemistry for anti-Flag M2 antibody in MCF-7 cells transfected with TNC-L (left image) and for TNC (Monoclonal, BC24) in MCF-7 transfected with vector control (right image). An average transfection efficiency of 35% was determined for each TNC isoform and staining confirmed that MCF-7 cells do not express TNC. (d) Western blot analysis for TNC in transiently transfected MCF-7 cells. This demonstrated a single species of TNC present in the whole cell lysate (WCL; i) and conditioned media (CM; ii) of transfected cells for each isoform. TNC-S is seen as a band at about 250 kDa, with slightly larger bands detected for TNC-16 and TNC 14/16, while TNC-L is detected at about 350 kDa.

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