Figure 4.

Gint4.T inhibits BM-MSC trans-differentiation into Cancer Associated Fibroblasts. (A) BM-MSCs were co-cultured with MDA-MB 231 or BT-549 cells for 5 days using a Boyden chamber containing inserts of polycarbonate membranes with 3 μm pores. α-SMA, FAP and FSP-1 mRNA levels were detected using Real Time-PCR. *P < 0.01,**P < 0.001 compared to BM-MSCs. (B) BM-MSCs incubated with 400 nmol/l Gint4.T or Scr were co-cultured with MDA-MB 231 or BT-549 cells as described above. α-SMA, FAP and FSP-1 mRNA levels were quantified using Real Time-PCR and reported as relative to Scr-treated cells, used as negative control. #P < 0.01, ##P < 0.001 compared to Scr aptamer. (C) Lysates from BM-MSCs grown for 48 hours in medium supplemented with 1% FBS or in CM from MDA-MB-231 (BM-MSC^{MDA-MB 231}) in the absence or in the presence of 400 nmol/l Gint4.T, were immunoblotted with anti-α-SMA and anti-vinculin antibodies. Values below the blot indicate signal levels relative to BM-MSCs grown in 1% FBS medium, arbitrarily set to 1 (labeled with asterisk). Molecular weights of indicated proteins are reported. (D) MDA-MB-231 cells were placed into upper matrigel-coated compartment of Boyden chamber and exposed to conditioned media from BM-MSCs grown as in (C) as chemoattractants (lower chamber) for 24 hours. Representative photographs of at least three different experiments are shown. The results are expressed as percent of invaded cells and reported as relative to BM-MSC CM.