Ab41478

Astrocytes were collected from subcultures and 2×10³ cells/well were seeded to 96 well plates (MTS assay) or 1×10⁴ cells/well in 6 well plates in DMEM/F-12 with 10% FCS. At confluence, before treatment cells were starved for 24 h in serum-free DMEM/F-12. Then medium was changed to serum-free DMEM/F-12 containing one or more of the fowling substances: 1 ng/ml active TGF-β2 (R&D Systems, 302-B2-010/CF), 250, 1000 or 2000 ng/ml human recombinant OPN (R&D Systems, 1433-OP-050/CF), 100 nM RGD peptide (Sigma, A8052), or an anti-CD44 blocking antibody (1∶100, Abcam, ab41478). In each experiment, control cultures were incubated with the solvent in serum-free medium alone.

Isolated or immunocaptured exosomes were tested for the presence of CD63, CD81, CD34, GAPDH, CD200, CD44 and CD105 using western blots as previously described [14] (link). Briefly, 10 µg of exosomes were lysed with Laemmli sample buffer (Bio-Rad Laboratories, Hercules, CA, USA), separated on 7–15% SDS/PAGE gels and transferred onto PVDF membrane (Millipore, Billerica, MA, USA) for western blot analysis. Membranes were incubated overnight at 4°C with antibodies specific for: CD63 (1∶200, sc-15363, Santa Cruz, CA, USA), CD34 (1∶2000, ab81289, Abcam, Cambridge, MA, USA), CD81 (1∶200, PA5-13582, Thermo Fisher, Pittsburgh, PA, USA), GAPDH (1∶500, FL-335, Santa Cruz, CA, USA), CD200 (1∶2000, AF2724, R&D, Minneapolis, MN, USA), CD44 (1∶1000, ab41478, Abcam, Cambridge, MA, USA), CD105 (1∶1000, ab169545, Abcam, Cambridge, MA, USA), platelet IIb/IIIa (1∶200, sc-73544, Santa Cruz, CA, USA). Next, the HRP-conjugated secondary antibody (1∶5000, Pierce, Thermo Fisher, Pittsburgh, PA, USA) was added for 1 hr at room temperature (RT) and blots were developed with ECL detection reagents (GE Healthcare Biosciences, Pittsburgh, PA, USA). The intensities of the bands on exposed films were quantified using Image J software (NIH, USA).

CD133 and BMI-1 proteins in OSCC tissues were detected with an Enhanced Chemiluminescence Western Blotting Detection Kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA) after binding of mouse anti-human CD133/1 (AC133) MAb (Miltenyi Biotec) and goat anti-human BMI-1 (sc30943, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Horseradish peroxidase (HPR)-conjugated anti-mouse and anti-goat secondary IgG antibodies were used (Santa Cruz Biotechnology). Anti-CD44 antibody (ab41478, Abcam, Cambridge, MA, USA), anti-CD1 (ab95587, Abcam), anti-Cdc2 (1:1,000 dilution, 9112S, Cell Signaling Technology, Danvers, MA, USA), and anti-GAPDH antibody (ab9485, Novus Biologicals, Littleton, CO, USA) were used for western blot analysis. Cycloheximide at a concentration of 10 μM was used for inhibition of protein synthesis in CA9-22 cell cultures. Catharidic acid (CA, CAS#28874-45-5, 1 μM) and arginine vasopressin (AVP, CAS#113-79-1, 10-6 mol) were used for inhibition of the Cdc2 protein and AVP protein, respectively, after cells were transfected with Id1+p65. CH, CA and AVP were all purchased from Sigma-Aldrich (St. Louis, MO, USA).