Sparc

To produce the lysate from tumor tissue, 100 mg tissue was lysed and homogenized in radio immuno-precipitation Assay (RIPA) buffer (Sigma, Australia) containing 10% protease inhibitor cocktail. The samples were then centrifuged at 10,000 g at 4° C for 10 minutes and the protein content in supernatant was quantified [17 (link)]. Fifty micrograms protein were fixed on 12% gels and electrophoresed for 2 hours at 85 V and transferred to Poly vinylidene Fluoride (PVDF) membranes. The membranes were incubated with the primary antibodies ( VEGF and SPARC, 1:200 dilutions, Santa Cruz Biotechnology) for overnight at 4°C followed by one hour incubation with secondary antibodies (anti-mouse from Santa Cruz Biotechnology and anti-rabbit from Cell Signalling, USA). The bands were visualized by an enhanced chemiluminescence detection kit (GE Healthcare, Australia). The blots were then stripped using Seppro western blot stripping buffer (Sigma, Australia) and re-probed with GAPDH (1:1000 dilution, Sigma, Australia).

Primary antibodies for EGFR (rabbit, Cell Signaling), NOTCH1 (rabbit, Cell Signaling), AKT (rabbit, Cell Signaling), PAX6 (rabbit, COVANCE), PTEN (A2B1, mouse, Santa Cruz), NES (mouse, EMD), Gamma Secretase (Ab kit for NCSTN), PSEN1 (rabbit, Cell Signaling), SPARC (mouse, Santa Cruz), AGAP2 (rabbit, Abnova), ANGTP1 (rabbit, Abnova), ANGTP2 (mouse, Abnova), CHIL31 (rabbit, Abnova), and EFEMP1 (rabbit, Abnova) were diluted 1:500 or 1000, and for GFAP (C-19, rabbit, Dako) and ACTB (IgM-specific mouse, Millipore) were diluted 1:10,000 for immunoblotting as described previously [11 (link)].
Gelatin zymography and Matrigel invasion assays were performed following procedures described previously [11 (link)]. For the invasion assay in this study, 5×10⁵ cells in 500 μl DMEM/F12 were loaded in 2-3 replications on Matrigel (1 μg/ml)-coated trans-well in 12-well plates (8μm; Fisher Scientific), and 1 ml DMEM/F12 was added to the bottom chamber. For 51B, addition of a small amount of bovine serum (final 0.05%) in the medium of the bottom chamber was applied to enhance migration of cells in penetration through the transwell-filter after degrading the Matrigel, but not to interfere cell-mediated matrix degradation by proteases within serum. Images of invasion were taken 48 hours later and cells were counted.

For immunofluorescence staining, cells were washed with PBS and fixed with 4% paraformaldehyde for 10 min, permeabilized with 1%Triton X-100 (Sigma) for 1 h, blocked with 5%BSA (Sigma) for 1 h at room temperature, then incubated with primary antibodies at 4 °C overnight and then incubated with secondary antibodies for 1 h at room temperature. The primary antibodies included Gata4 (Santa Cruz Biotechnology, Dallas, TX, USA), Gata6 (Cell Signaling Technology, Danvers, MA, USA), Sox17 (Cell Signaling Technology), Sall4 (Santa Cruz Biotechnology), Oct4 (Santa Cruz Biotechnology), Sox2 (Calbiochem, Billerica, MA, USA), Nanog (Santa Cruz Biotechnology), Cdx2 (Bio Genex, San Ramon, CA, USA), Sparc (Santa Cruz Biotechnology), Cdh1 (Abcam, Waltham, MA, USA), Desmin (Millipore), Cytokeratin (Millipore), and βIII-Tubulin (Abcam). Secondary antibodies include goat anti-mouse IgG Alexa Fluor 546 (Thermo Fisher Scientific, Waltham, MA, USA), goat anti-mouse IgG Alexa Fluor 488 (Thermo Fisher Scientific), goat anti-rabbit IgG Alexa Fluor 546 (Thermo Fisher Scientific), and goat anti-rabbit IgG Alexa Fluor 488 (Thermo Fisher Scientific). The nuclei were stained with DAPI (Sigma).