Figure 5.

Assessment of UGDH expression, impact of knocking down UGDH levels on HA synthesis, and determining the consequences of Slit2 on UGDH expression. (A) Confluent fibrocyte and GD-OF monolayers were harvested, RNA extracted, reverse-transcribed, and subjected to RT-PCR for UGDH mRNA. Values were normalized to their respective GAPDH levels. (B) GD-OF were transfected with scrambled (control) (3 µg) or UGDH-targeting siRNA (3 µg), re-cultured for 3 days, and media collected and subjected to HA ELISA. Cell layers were collected and protein levels determined. (C) GD-OF were transfected with scrambled (3 µg) or Slit2-targeting siRNA (3 µg), re-cultured, and assessed for UGDH mRNA levels by RT-PCR. (D) Fibrocytes were incubated with nothing or rhSlit2 (50 ng/ml) for 7 to 9 days. Cell layers were processed as in panel (C). Data are expressed as the mean ± SD; * P < 0.05; **; P < 0.01 of 3 independent determinations. Experiments were performed 3 times.