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Fig. 4. Recombinant heparanase increases branching morphogenesis of the intact
SMG, increases phosphorylation of ERK1/2, and when added to isolated
epithelium cultured in a 3D ECM, increases lateral branching, end bud clefting
and duct elongation. (A) E12 SMGs were cultured with 5 µg/ml of
either inactive (I), active (A), or unprocessed (U) forms of heparanase for 48
hours. (B) The number of buds was expressed as a ratio of the number of
buds at 48 hours/number of buds at 1 hour (T48/T1). (C) Western blot
analysis of phospho-ERK1/2 and total ERK1/2 after 48 hours of treatment with
either inactive, active, or unprocessed heparanase resulted in an 
3-fold
increase in pERK1/2 with active and an 2.3-fold increase with unprocessed
heparanase. (D) Isolated SMG epithelia were cultured with 200 ng/ml of
FGF10 (a sub-optimal dose for growth) and treated with 5 µg/ml of either
inactive (which appeared similar to a carrier control, not shown), active or
unprocessed heparanase and compared after 48 hours with epithelia cultured
with 500 ng/ml of FGF10. The total number of end buds was counted from at
least five glands/condition and the experiments repeated twice. ANOVA compared
with inactive heparanase; *P<0.05;
**P<0.01.
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