Beclin 1

Protein expression levels of LC3B, Beclin1 and CXCR4 in the UC‐MSCs and of CXCL12 (Cell Signaling Technology, Danvers, MA) in the liver tissues were detected by Western blot assays. Total protein was extracted from the cells and tissues using lysis buffer (Kaiji). LC3B, Beclin1, CXCR4, CXCL12 and GAPDH were measured and analysed as previously described.7 The following first antibodies were used: LC3B‐, Beclin1, CXCR4, CXCL12, and GAPDH (1:1000; from Cell Signalling Technology), and the secondary antibody was used: (anti‐rabbit IgG) (1:5000; Sigma‐Aldrich).

The animals (n = 5 for each experimental group) were sacrificed by decapitation, and the brains were quickly removed. The cerebral cortices obtained from the 3xTg-AD mice and sham- and post-ischemic rats were dissected and frozen at -80°C until further analysis. The samples were homogenized in lysis buffer containing 150 mM NaCl, 20 mM Tris (pH 7.4), 10% glycerol, 1 mM EDTA, 1% NP40, 100 μM phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin and leupeptin (Sigma), and 100 μM orthovanadate. The lysates were clarified by centrifugation at 14,000 rpm for 5 min. A protein assay was performed, and 250 μg of protein was incubated overnight at 4°C in the presence of Beclin-1 or Vps34 antibodies (1:250, Cell Signaling Technology). Protein G-sepharose beads were added, and the samples were incubated for an additional 2 h at RT. The immune complexes were washed three times using an immunoprecipitation lysis buffer prior to the SDS-PAGE and immunoblotting. The proteins were separated using 10% SDS-PAGE, transferred onto nitrocellulose membranes (Amersham), and probed with Beclin-1 (1:1,000; Cell Signaling) or Vps34 antibodies (1:500; Calbiochem). The lysates were used as the positive controls, and incubation with IgG peptide was used as the negative immunoprecipitation control.

Human colon cancer cells HT-29 and Caco-2 were maintained in DMEM (Hyclone, Cramlington, UK) supplemented with 10% fetal bovine serum (FBS) (Hyclone, Cramlington, UK), 100 U/ml penicillin/streptomycin (Hyclone, Cramlington, UK). MG-132 was obtained from Cell Signaling, 3-Methyl adenine (3-MA) from Millipore (Hayward, CA, USA) and Chloroquine (CQ) from Sigma-Aldrich (Saint-Quentin Fallavier, France). Antibodies against LC3, AKT, Beclin-1, Ubiquitin, mTOR, phospho-mTOR (Ser 2448), caspase-3, caspase-7, and p53 were obtained from Cell Signaling (USA); those against cleaved caspase 3, and phospho-AKT (Ser 473) were obtained from Millipore (Millipore, Hayward, CA, USA), and those against p62/SQSTMI and cleaved PARP were obtained from Abcam (Abcam, Cambridge, UK). Antibody against PARP (full-length and cleaved) was purchased from BD Pharmingen. Antibody against β-actin was obtained from Santa Cruz Biotechnology, Inc (USA). Beclin-1 and control siRNA were purchased from Cell Signaling.