Figure 8.
Heparanase is expressed by HEK293T and HSG cells and is required for lacritin-dependent mitogenesis. (A) Lysates of HSG cells (lane 1) and HEK293T cells stably expressing human SDC1 (lanes 2) versus 2 M NaCl eluant of each after incubation with HiTrap heparin affinity columns (lanes 3 and 4, respectively). Blotting is with polyclonal anti–human heparanase-1 (HPSE1) antibody. (B) Lysates from HSG cells that had been mock transfected or transfected with 1 nM heparanase-1 siRNA. Blotting is with polyclonal anti–human HPSE1 or anti-tubulin antibodies. (C) Proliferation assay in which HSG cells were treated with 10 nM lacritin or 1 nM EGF 48 h after being mock transfected or transfected with 10 nM of Ambion's negative control siRNA #1 (neg), 1–100 nM HPSE1 siRNA, or 1 nM HPSE2 siRNA. Some HPSE1 siRNA cells were lacritin treated for 24 h in the presence of 1 μg of heparanase-enriched eluant (A) from HEK293T cells stably expressing SDC1 (1 nM + HPSE) or 0.0001 U of bacterial heparitinase. Error bars indicate SEM. (D) Sepharose CL-6B gel filtration chromatography of HS from lacritin and FGF2 affinity enriched SDC1 isolated from normal or HPSE1-depleted HSG cells. Lysates from cells labeled with 50 μCi/ml Na2 35SO4 in DME for 48 h were affinity precipitated with FGF2-GST or lacritin-intein. Equal microgram amounts of SDC1 bound to beads were digested with chondroitin ABC lyase to remove CS, eluted with 2 M NaCl, and subjected to NaBH4 eliminative cleavage. Released HS was neutralized by drop-wise addition of 1 M HCl and subjected to Sepharose CL-6B gel filtration chromatography to compare the relative size of HS chains. V0, void volume (dextran blue); Vt, total volume (sodium dichromate).