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.