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Suppl. Figure legends
Suppl. Figure 1. Inducible expression of heparanase, 8C and T5. A. Enzymatic activity. CAG myeloma cells were infected with Tet-inducible heparanase, 8C, or T5 gene constructs. Cells were left untreated (-) or incubated with Dox (1μg/ml; +) for 24 h. Cell lysates were then prepared and heparanase activity was evaluated as described in 'Materials and Methods'. B. Colony formation following heparanase, 8C, and T5 induction. CAG cells (2x103) infected with inducible heparanase, 8C, or T5 gene constructs were suspended in soft agar and grown for 5 weeks without or with Dox (+Dox). Representing dishes (left panels) and colonies (right panels) are shown. Colony number was quantified and presented as fold increase in colony number in the presence of Dox (C). D. Immunoblotting. CAG-heparanase cells were cultured in the absence (0) or presence of Dox for 8, 16, and 24 hours. Medium (Med) and lysate (Ly) samples from each time point were subjected to immunoblotting applying anti heparanase (upper and second panels), anti actin (third panel), anti phospho Src (pSrc, fourth panel) and anti Src (lower panel) antibodies.
Suppl. Figure 2. A. Heparanase gene silencing. CAG cells were infected with anti-heparanase shRNA (sh-Hepa) or control shRNA (sh-Con) and heparanase expression was quantified by real time PCR. B, C, H. CXCL10 over-expression. CAG clone 13 (B), RPMI-8266 (C) and CAG-heparanase cells (H) myeloma cells were infected with CXCL10 or control empty vector (Vo) and CXCL10 expression was quantified by real time PCR and presented as fold increase compared with control cells. D. Colony formation. CAG-heparanase cells were infected with CXCL10 or control empty vector (Vo) and were seeded (2x103/35 mm dish) in soft agar and were grown for three weeks. The number of colonies developed by cells over expressing CXCL10 is shown graphically related to the number of colonies developed by control (Vo) cells. E. CXCL10 gene silencing. CAG cells were infected with control (sh-Con) or anti-CXCL10 shRNA (sh-CXCL10) and CXCL10 expression was quantified by real-time PCR. F, G. Anti-CXCL10 neutralizing antibody. RPMI-8266 (F) and U266 (G) myeloma cells were cultured in the presence mouse IgG (Mo. IgG) or anti-CXCL10 neutralizing antibody (anti-CXCL10; 20 µg/ml) for three days and cell proliferation was evaluated by MTT assay as described under 'Material and Methods'.
Suppl. Figure 3. A. Immunohistochemical analysis. Paraffin-embedded 5 micron sections of tumor xenografts produced by CAG-heparanase control (Vo) and CXCL10 over expressing cells were stained with anti-CXCL10 antibody. B. CXCL10-Ig purification. Medium conditioned by Chinese hamster ovary DG44 cells over expressing CXCL10-Ig was applied onto protein-A-Sepharose column and bound material was eluted with citric acid, pH 2.5. CXCL10-Ig appears as a double band with apparent molecular weight of ~36 kDa under reducing conditions (+), and 72 kDa double band under non-reducing conditions (-), suggesting that the fusion protein exists as a dimer in solution. C. CXCL10-Ig activity. Migration of T-cells was evaluated in the absence (cells alone) or presence of CXCL10, CXCL10-IgG, CXCL10-IgG + anti CXCL10 neutralizing antibody, or β-actin-IgG fusion protein. The CXCL10-IgG appear as active as CXCL10 itself, with the advantage of higher stability and extended half-life in vivo. D. Immunohistochemistry. CAG myeloma cells were infected with Tet-inducible heparanase and inoculated (1x106) subcutaneously into SCID mice (n=6). Paraffin-embedded 5 micron sections of tumor xenografts produced in the absence or presence of Dox were stained with anti-CXCL10 antibody. Note decreased CXCL10 staining upon heparanase induction.
Suppl. Figure 4. A-C. CXCL10 repression does not require heparanase enzymatic activity. A. Tet-on CAG-heparanase cells were cultured in the absence (-) or presence of Dox (+) without or with heparin or SST0001 (50 μg/ml) for 24 hours and CXCL10 expression was quantified by real-time PCR. Note reduced CXCL10 expression in the presence of inhibitors of heparanase enzymatic activity. CXCL10 repression by heparanase C-terminal domain (8C) and T5. CAG cells were infected with Tet-inducible 8C (B) or T5 (C) gene constructs and were left untreated (-) or incubated with Dox (1 μg/ml; +). Total RNA was extracted at the time indicted and CXCL10 expression was quantified by real time PCR. D. Control (Vo) and CXCL10-infected CAG myeloma cell lysates were subjected to immunoblotting applying anti-cleaved caspase 3 (upper panel) and caspase 3 (second panel) antibodies. The cells were similarly subjected to immunostaining applying anti-cleaved caspase 3 antibody (third panels). Percent cleaved caspase 3-postive cells are shown graphically in the lower panel. E. Serum-starved U266 myeloma cells were incubated without (-) or with latent heparanase (+, 1µg/ml) for 60 min. Cell lysates were then prepared and subjected to immunoblotting applying anti-phospho-Erk (left upper panel), anti-Erk (left middle panel), anti-phospho-STAT3 (right upper panel), and anti-STAT3 (right middle panel) antibodies. Erk and STAT3 phosphorylation following addition of heparanase was quantified by densitometry analyses and in shown graphically in the lower panels as fold-increase compared with control cells.