Effect of addition of exogenous HA and hyaluronidase treatment.

<p>Confluent TM cells were treated for 2 days with 1 mM 4MU and then HA of different molecular weights (high MW = 1.5 MDa; medium MW = 700 kDa; low MW = 40 kDa) was exogenously added for a further 3 days. Versican and fibronectin protein levels in conditioned media were assessed by Western immunoblot. (A) Versican V1 isoform and (B) fibronectin protein levels are significantly decreased by 4MU treatment in all cases, but addition of 3 concentrations (100 µg/ml, 500 µg/ml and 1 mg/ml) of HA of HMW, MMW and LMW did not significantly alter levels compared to 4MU treatment alone. The Western immunoblots show data from a 500 µg/ml treatment. All lanes were from adjacent lanes of the same immunoblot. Error bars are the s.e.m.; n = 3. (C) Hyaluronidase (1.5 µg/ml; 1 unit) was added to confluent TM cells in serum-free media each day for 3 days. Western immunoblots and densitometry showed that versican V1 and fibronectin protein levels were significantly decreased by hyaluronidase treatment. All lanes were from adjacent lanes of a single immunoblot. Versican and fibronectin values were normalized to total protein loaded and are presented as a percentage of the control. Error bars are the s.e.m.; n = 4; * p = 0.03 and ** p = 0.001 by one-way ANOVA.