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. 2013 Jun 4;8(6):e66281. doi: 10.1371/journal.pone.0066281

Figure 1. LOX mRNA and protein are downregulated by EWS/FLI1 in the A673 Ewing sarcoma cell line.

Figure 1

A) A673/TR/shEF cells were stimulated by different periods of time with doxycycline (DOX, 1 µg/ml) to induce the expression of an EWS/FLI1-specific shRNA. The levels of LOX, EWS/FLI1 and TBP (reference gene) mRNA were quantified by multiplex real time qRT-PCR. For each time point, LOX and EWS/FLI1 mRNA levels were normalized to that of TBP and referred to unstimulated cells. The figure shows the data (mean±SD) of one out of two independent experiments done in triplicate with equivalent results. B) LOX protein levels were also determined by western-blot in the A673/TR/shEF cells stimulated with doxycycline. The same blot was stripped and successively incubated with anti-FLI1 antibody to assess the expression of EWS/FLI1 and with anti-α-tubulin as a control for loading and transferring. LOX mRNA and protein levels were strongly downregulated by EWS/FLI1. C) LOX protein levels were determined by western-blot in 8 Ewing derived cell lines and in normal fibroblasts IMR-90, used here as a positive control of LOX expression. A673/TR/shEF cells stimulated with doxycycline for 48 hours were also included. The type of EWS/FLI1 (EF; EWS exon/FLI1 exon) and EWS/ERG fusion (EE; EWS exon/ERG exon) present in each Ewing cell line are indicated. LOX protein expression was nearly undetectable in Ewing derived cell lines. D) LOX mRNA levels were quantified by qRT-PCR in a set of Ewing's primary tumors (n = 21). Normal fibroblasts IMR-90 are used as a positive control. mRNA levels were normalized to that of TBP (mean±SD). LOX mRNA levels observed in Ewing tumors were very low compared to those observed in the control fibroblast cell line IMR-90.