Figure 4

Glycosaminoglycans profile of SKBR3, MCF7 and MCF7-HPSE1 cells. Sixty percent confluent cells were incubated for 18 hours in serum-free medium containing 150 mCi/ml [³⁵S]-sulphate. Protein-free GAG chains were prepared from the cells and culture medium by incubation with maxatase. Aliquots from the medium and cells were submitted to agarose gel electrophoresis (0.05 M diaminopropane acetate buffer, pH 9.0) and the sulphated GAG identified and quantified as described in methods. (A), Heparan sulfate (HS) and dermatan sulfate (DS) from SKBR3; (B), HS and chondroitin sulfate (CS) from MCF7; (C), HS and DS from MCF7-HPSE1. *P < 0.05, compared to the respective fraction of MCF7 cells, **P < 0.05, medium versus cellular fraction of the same cell lineage. (D), mRNA expression of DS glucuronosil C5 epimerase in MCF7 versus MCF7-HPSE1 cells by qRT-PCR. The RNA was extracted using TRIzol reagent, converted into cDNA by RT-PCR and submitted to qRT-PCR using specific primers for glucuronosil C5 epimerase. The values were corrected by RPL13a (ribosomal protein) and GAPDH expressions. Each bar indicates the mean ± SD of triplicate assays. *P < 0.001.