Supplementary Figure Legends
Supplementary Figure S1. Heparanase enhances autophagy. A. Control (Mock) and heparanase over expressing (Hepa) rat C6 glioma cells were cultured under serum-free conditions without (Con) or with chloroquine (Chl) for 3h. Cell lysates were then subjected to immunoblotting applying anti LC3 (upper panel) and anti-actin (lower panel) antibodies. B. Control (Mock, upper panel) and heparanase (Hepa, lower panel) over expressing FaDu cells were fixed with glutaraldehyde and subjected for EM analysis. Shown are representative images at x10,000 magnification. Autophagy quantification. EM images of control (Mock) and heparanase over expressing (Hepa) FaDu cells not deprived (Con) or deprived of amino acids plus chloroquine (AA+Chl) were counted for autophagic vacuoles in at least 12 cells in each treatment condition. Average number of autophagic vacuoles is presented graphically ±SE (B, right panel). C. Treatment with PG545 results in decreased intracellular content of heparanase in vesicles. Heparanase over expressing U87 cells were cultured without (Con) or with PG545 (PG, 25 µg/ml) for 24h. Cells were then fixed with 4% PFA and subjected to immunofluorescent staining applying anti-heparanase antibody. Heparanase positive vesicles were quantified in 12 cells and are presented graphically as average ± SE. D. Immunostaining. Five micron sections of U87 tumor xenografts untreated (Con) or treated with chloroquine (Chl), PG545 (PG) or both were subjected to immunostaining applying anti-phospho-p70 S6-kinase. Note increased P70 S6-K phosphorylation following autophagy and heparanase inhibition. E. Control (Mock) and heparanase over-expressing (Hepa) U87 glioma cells were deprived of amino acids for 3h and cell morphology was evaluated by light microscopy. Shown are representative photomicrographs at x40 magnification.
Supplementary Figure S2. Heparanase provide cells with chemo-resistance. A. Chemoresistance. Control (Mock) and heparanase over expressing (Hepa) U87 glioma (upper and lower panels) and FaDu pharyngeal carcinoma (middle panel) cells were cultured without (0) or with the indicated concentration of cisplatin or doxorubicin and cell viability was evaluated by MTT assay as described under 'Materials and Methods'. Note resistance of cells over expressing heparanase to chemotherapeutics. B. Reduced phospho-p70 S6-kinase. Lysate samples of control (Mock) and heparanase over expressing (Hepa) FaDu cells were subjected to immunoblotting applying anti-phospho-p70 S6-kinase (p-p70; upper panels), anti-p70 S6-kinase (p70; second panels), and anti-actin (lower panels) antibodies. C. Hpa-KO MEF are more sensitive to mTOR inhibitor. MEF obtained from control (Con) and heparanase-KO mice were incubated without (WO) or with Torin (mTOR inhibitor; 500 µM, 3h) and cell lysate samples were then subjected to immunoblotting applying anti-phospho-p70 S6-kinase antibody. The level of p70 S6-kinase phosphorylation after Torin treatment is presented graphically as percent of untreated cells. Note that Hpa-KO MEF are more sensitive to mTOR inhibitor. D. Cell invasion. MEF obtained from control (Con) and Hpa-KO mice (2x105) were plated on Matrigel-coated inserts without or with chloroquine (50 µg/ml). Cells invading the reconstituted basement membrane were counted after 18 h. Note that Hpa-KO cells are less invasive and are more sensitive to chloroquine than control cells.
Supplementary Figure S3. PTEN and TSC do not localizes to acidic vesicles. A. Control (Mock) and heparanase (Hepa) over expressing U87 cells were incubated with LysoTracker (75 nM, 2h, 37oC; middle panels, red), fixed in 4% PFA and subjected to immunofluorescent staining applying anti-heparanase antibody (upper panel, green). Merged images are shown in the lower panels. B-C. Control and heparanase over expressing cells were similarly stained with LysoTracker (middle panels, red) and anti-TSC1 (B) or anti-PTEN (C) antibodies (upper panels, green). Nuclei counterstaining appears in blue. Merged images are shown in the lower panels. Note that heparanase localizes with LysoTracker-positive acidic vesicles, but no such co-localization is evident for TSC1 or PTEN.