Amd3100

Prior to the western blot analysis and migration assay, the cardiomyocytes were pretreated with or without PI3K inhibitor (LY294002; 20 μm/l; Cell Signaling Technology, Inc., Danvers, MA, USA) for 2 h prior to treatment with Ex-4 (20 nm/l) for 24 h. Following treatment, the conditioned medium was collected and used for subsequent experiments. In addition, prior to western blotting and migration assay, the ADSCs were incubated with or without either LY294002 (30 μm/l) for 2 h or an SDF-1α/CXCR4 cascade antagonist (AMD3100; Abcam; 5 μg/ml) (23 (link)) for 1 h, prior to treatment with Ex-4 (20 nm/l) for 24 h.

To investigate the role of the PI3K/Akt pathway in Ex-4-mediated MSC proliferation, migration and survival, LY294002 (20 μm/L, Cell Signaling Technology) was added to the MSC medium for 4 h before Ex-4 treatment to block PI3K/Akt pathway activation. For the proliferation experiments (cell cycle analysis and EdU assay), MSC were treated with Ex-4 for 24 h before the above assays. For the chemotaxis assay, MSC were cultured with Ex-4 for 24 h and then seeded in chemotaxis chambers for 12 h. Moreover, to block the interaction between CXCR4 and SDF-1α, MSC were incubated with or without CXCR4 antibody (AMD3100, Abcam, USA, 10 μg/ml) for 2 h before Ex-4 treatment (20 nm/L). In the H₂O₂-induced apoptosis model, MSC were pretreated with Ex-4 (0–20 nm/L) for 12 h and then incubated with 0.3 mM H₂O₂ for 12 h.

Synthetic chemokines (CXCL14, CCL2, CCL5, full-length CXCL12α, and CXCL12α N-terminal peptide consisting of amino acid residues 1–9) were chemically synthesized, as previously described (40 (link)). Human CXCL14 and murine CCL1—both conjugated to the fluorochrome Alexa Fluor 647 attached to a C-terminal lysine residue—were purchased from Almac Sciences (Edinburgh, United Kingdom). For NMR study, ¹⁵N-labeled CXCL12α was produced recombinantly and purified as previously described (41 (link)). The CXCR4 antagonist AMD3100 was purchased from Abcam (Cambridge, United Kingdom).

In the BMSC experiment, the control groups' BMSCs were cultured for 15 min in osteogenic inductive media. In the experimental groups, BMSCs were treated with 100 ng/ml SDF-1(Sigma, S5816) for 15 min with or without 2 hr pretreatment of 200 ng/ml AMD3100 (Abcam, ab120718) in osteogenic inductive media. In the HUVEC experiment, HUVECs were treated with 20 or 50 ng/ml SDF-1 for 30 min with or without 2 hr pretreatment of 200 ng/ml AMD3100.
After washing by phosphate buffer solution (PBS) for three times, protein from cells were extracted. An equal amount of proteins (30 μg) was loaded onto 10% Tris/glycine gels for electrophoresis and transferred to a PVDF membrane. 5% nonfat milk was used to block for 1 hour at room temperature with shaking. Primary antibodies, anti-p-ERK (Affinity Bioscience, AF1015), anti-total Erk1/2 (Selleck, A5029), anti-p-Akt (HuaAn，ET1607-73), and anti-total AKT (A5023; Bimake), were, respectively, added to incubate at 4°C overnight. Then, TBST was used to wash the membrane for 3 times, each time for 10 minutes. Subsequently, the membrane was incubated with horseradish peroxidase-linked secondary antibodies. TBST was used to wash the membrane 3 times for 10 min each time again.

Recombinant mouse TFF2 (#RPA748MU01) was purchased from Uscn Life Science (Texas, USA). AMD3100 (#AB120718) was purchased from Abcam (Cambridge, GBD).