Figure 1. Effect of norrin on tPA and uPA
expression, and on survival of RGC-5 cells. In the absence (A,B,E)
or presence of 2.0 μM staurosporine (SS; C,D,F), RGC-5 cells
were left untreated or treated for 48 h with varying concentrations of
norrin (n=3 experiments). At the end of 48 h, conditioned medium was
collected and proteolytic activity of tissue plasminogen activator
(tPA) and and urokinase plasminogen activator (uPA) was determined by
zymography assays (A,C), relative amount of proteolytic activity
was determined by densitometric analysis (B,D), and cell
viability was assessed by
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)
assays (E, F). In the absence of SS, norrin had no effect on low
levels of uPA expressed constitutively by RGC-5 cells (A,B; NS
is not significant). In addition, lower concentrations of norrin had no
effect on cell survival, but higher concentrations of norrin (50 and
100 ng/ml) increased cell survival significantly (E,
*p<0.05). Compared to low levels of uPA expressed by untreated
cells, uPA (D, #p<0.05) and tPA (D, **p<0.05)
levels were significantly increased in SS alone or in SS and
norrin-treated cells. Nonetheless, survival of RGC-5 cells decreased
significantly in the presence of SS alone (F, *p<0.05), but
not in the presence of SS and norrin (F, **p<0.05).