Endothelin 1

Female immunodeficient mice (Laboratory Animal Center, Academy of Military Medical Sciences, Beijing, China) at the age of 8 weeks with an average weight of approximately 20 g were used in this experiment. Procedures were carried out with approval from the Animal Use and Care Committee of Beijing Institute of Radiation Medicine (Permit Number: 2012005). Mice were diveded into three groups (n = 8 per group). Cell transplantations were performed as previously described [19] (link). Either MDA-MB-231 (2×10⁶ cells) alone or mixed with an equal number of H₂O₂-induced MSCs or passage 5 MSCs were subcutaneously injected in the right flank region of the animals. Tumors were measured every 7 days using a digital vernier caliper, and the tumor volume was calculated as (length×width²)/2. On day 42, mice were sacrificed by cervical dislocation humanely. Subcutaneous tumors werev removed and weighed before they were fixed in 4% paraformaldehyde for histological examination. The immunohistochemical experiment for endothelin-1 (1∶100, Abcam) was executed as previously described [19] (link). Blood vessels were counted under 100× magnification.

Cardiac tissues samples were homogenized with lysis buffer and diluted 1:1 with 2 × SDS sample buffer (Invitrogen Novex, Carlsbad, CA). An equal amount of protein (30 μg) was loaded onto each lane of an 8% to 16% Tris‐Glycin gel (Helixx). Proteins were separated by electrophoresis and transferred to a nitrocellulose membrane using an electro‐blotting apparatus (Invitrogen, Carlsbad, CA). Membranes were incubated with 5% Bovine Serum Albumin (BSA) for 1 h to decrease nonspecific binding. Samples were then incubated with the following primary antibodies overnight at 4°C: Connective Tissue Growth Factor (CTGF) (Abcam, Cambridge, UK), SMAD3 and 4 (R&D Systems Inc. Minneapolis, MN), pSMAD3 (Cell Signaling Techonology Inc. Danvers, MA) Endothelin‐1 (Abcam, Cambridge, UK), and matrix metalloproteinases 2 (MMP‐2) (EMD Millipore, Billerica, MA). Caspase 3 and 8 (Cell Signaling Techonology Inc. Danvers, MA), α and β MHC (EMD Millipore, Billerica, MA) samples were washed and incubated with peroxidase – conjugated secondary antibody, and detected using the Amersham ECL (GE life sciences, Buckinghamshire, UK) detection kit. Glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) was used as the internal standard.