Electrochemiluminescence kit

Total protein was extracted with radioimmunoprecipitation assay lysis buffer (cat. no., 89900, Thermo Scientific) with protease and phosphatase inhibitors (Roche Applied Science, Penzberg, Germany). Total proteins were quantified using a BCA kit (Pierce; Thermo Fisher Scientific, Inc.; cat. no., 23225) and subjected to 12% SDS/PAGE gel with 50 µg in each lane, followed by transfer onto polyvinylidene fluoride membranes (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Membranes were blocked with 5% nonfat milk in TBST (100 mmol/l Tris-HCl, 1% Tween 20, pH 7.5) for 1 h at room temperature, and then incubated with appropriate aforementioned primary antibodies in 1:2,000 dilution at 4°C overnight. Signals were detected using the aforementioned horseradish peroxidase-conjugated secondary antibody in 1:5,000 dilution (Thermo Scientific) and an electrochemiluminescence kit (cat. no., RPN2109; Amersham; GE Healthcare Life Sciences, Uppsala, Sweden). The quantitative analysis was performed using Tanon image version 1.0 software (Tanon Science and Technology, Co., Ltd., Shanghai, China).

ASCs from the 4 sample groups were cultured for 2 weeks at 37°C with 90% humidity. The cells were lysed in lysis buffer [150 mM NaCl, 50 mM Tris-HCl (pH 8.0), 0.1% SDS, 1% Triton X-100] containing protease and phosphatase inhibitors (Roche Diagnostics, Basel, Switzerland). Cell lysate protein content was determined using a bicinchoninic acid protein assay kit (Sigma-Aldrich; Merck KGaA). An equal amount of whole cell extracted protein (10 µg) was subjected to 12% SDS-PAGE gel, transferred to PVDF membranes and blocked by non-fat milk in an incubator (26°C, 40 × g, 2 to 4 h). The blocking mixture was then discarded, and a hybrid solution containing primary antibodies (GATA4, Nkx2.5, cTnT, desmin, Cx43, MyoD, α-SMA) was added and incubated at 4°C overnight. A secondary antibody hybrid solution (1:10,000) was added the following day, and membranes were incubated at 26°C for 1 h (40 × g). An electrochemiluminescence kit (Thermo Fisher Scientific, Inc.) and Kodak gel imaging system 2200 (Kodak, Rochester, NY, USA) were used to collect and analyze images.

Paclitaxel was purchased from Bristol-Myers Squibb (New York, NY, USA). A Cell Counting Kit-8 (CCK-8) was obtained from Dojindo Molecular Technologies (Kumamoto, Japan). The Protease Inhibitor Cocktail was provided by Roche (Basel, Switzerland). Mouse anti-human COX4 antibody was obtained from Molecular Probes (Eugene, OR, USA). Goat anti-human lamin B and mimitin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse anti-human flotillin-1 and rabbit anti-human β-actin antibodies were obtained from eBioscience (San Diego, CA, USA). Rabbit anti-human 14-3-3 ζ/δ antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA) and Sigma-Aldrich (St. Louis, MO, USA), respectively. The electrochemiluminescence kit was purchased from Thermo Scientific (Boston, MA, USA).

After removal of the brain, brain tissues around the infarcted region were removed and cut into pieces. The brain tissue was subsequently homogenized, lysed using a protein isolation kit (GE Healthcare Life Sciences) and centrifuged (11,000 × g at 4°C). The bicinchoninic acid (BCA) method was used to determine the total protein concentration. Western blotting was performed according to standard procedures (21 (link)). Briefly, protein samples (25 µg) obtained from each group were run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12%) and transferred onto nitrocellulose membranes for western blot analysis. Subsequently, the membranes were blocked in 5% skim milk for 2 h in room temperature. The membranes were subsequently incubated with the following primary antibodies: Rabbit anti-GRP78 (1:1,000), rabbit anti-CASP12 (1:1,000), and rabbit anti-CHOP (1:1,000) for 60 min at room temperature. The nitrocellulose membranes were washed thrice, and incubated with secondary antibody (HRP-labeled goat anti-rabbit IgG; cat. no. A16104; Thermo Fisher Scientific, Inc.) at 4°C for 2 h. The staining of the blots was enhanced using an electrochemiluminescence kit (Thermo Fisher Scientific, Inc.). The densities of the blots were quantified using the Quantity One software (v4.62; Bio-Rad Laboratories, Inc.).

At 24 h post-treatment with Huc-MSCs-exo, protein was extracted from cells using the triplePrep kit (cat. no. 28-9425-44; ReadyPrep; GE Healthcare Life Sciences). The protein levels were quantified using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology), according to the manufacturer's instructions. A total of 25 µg/lane protein was separated via SDS-PAGE on a 10% gel and transferred onto nitrocellulose membranes. The membranes were blocked using 5% skimmed milk at room temperature for 2 h and incubated overnight at 4°C with the following primary antibodies: Rabbit polyclonal anti-E-cadherin (1:1,500; cat. no. AF0131; Affinity Biosciences), anti-GAPDH (1:1,000; cat. no. AG019; Beyotime Institute of Biotechnology), anti-Vimentin and anti-N-cadherin (1:3,000 and 1:1,000, respectively; cat. nos. ab92547 and ab76057, respectively; both Abcam). After washing with 0.1 M PBS, the membranes were incubated with the secondary antibody (HRP-labeled goat anti-rabbit IgG; cat. no. A16104; Thermo Fisher Scientific, Inc.) at 4°C for 2 h. The blots were visualized using an electrochemiluminescence kit (Thermo Fisher Scientific, Inc.). The densities of the blots were quantified using the Quantity One software (version 4.62; Bio-Rad Laboratories, Inc.).