Sc 56053

Western blot analyses were performed as described in a previous study [53 (link)]. The protein concentrations of cell or tissue lysates were measured using the Lowry assay. Depending on the molecular weight of the target protein, 10 μg protein samples were separated using 8% or 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and electroblotted onto a nitrocellulose membrane. The membranes were blocked with 5% non-fat dry milk in Tris buffered saline plus Tween (TBST), immunoblotted with specific primary antibodies against CAV-1 (GTX79350, GeneTex, Irvine, CA, USA), eNOS (A15075, ABclonal, Woburn, MA, USA), BNP (1:500, sc-18813, Santa Cruz Biotechnology, Santa Cruz, CA, USA), cytochrome c (1:1000, sc-13156, Santa Cruz), caspase 3 (1:1000, sc-56053, Santa Cruz), and β-actin (1:1000, T5168, GeneTex), and detected using horseradish peroxidase conjugated secondary antibodies. Fluorography with an enhanced detection kit was used to visualize the signals (ECL, GE Healthcare Life Sciences, Buckinghamshire Amersham Pharmacia International).

The PLK1 inhibitor, volasertib, was purchased from Selleck Chemicals (Houston, TX, USA). Antibodies against PLK1 (4513), Bcl‐2 (2876), Bcl‐xl (2762), cleaved caspase‐3 (9664), PARP (9542), CD44 (3570), CD133 (64326), NANOG (4903), Cyclin B1 (4135), phospho‐Bad (9295), Bad (9292) and GAPDH (5174) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against FoxM1 (sc‐376 471) and caspase‐3 (sc‐56 053) were purchased from Santa Cruz Biotechnology, Inc. Cyclin D1 (ab139260) was obtained from Abcam (Cambridge, MA, USA). Phospho‐CDK1/CDC2 (orb127843) was purchased from Biorbyt Ltd (Cambridge, UK).

Parts of the heart were fixed with 4% paraformaldehyde and then wax embedded and sectioned (5 μm thick) for hematoxylin and eosin staining (H&E, Invitrogen, Carlsbad, CA, USA), as previously described [54 (link)]. The lipid content was quantified by Oil Red O staining (Bio Vision, Catalog # K58024, Mountain View, CA, USA). After staining with hematoxylin and washing with dH₂O, the section samples were washed and treated with 60% isopropanol for 5 min each time with gentle rocking. Thereafter, the section samples were extracted after Oil Red O staining with 100% isopropanol for 5 min with gentle rocking. Immunohistochemical staining was used to measure autophagic or apoptotic protein expression, with tissue sections (5 μm thick) incubated in blocking buffer (0.5% BSA, 0.05% Tween 20, and PBS) for 1 h at room temperature, followed by specific primary antibodies against BNP (1:100, sc-271185, Santa Cruz), cytochrome c (1:100, sc-13156, Santa Cruz), and caspase 3 (1:100, sc-56053, Santa Cruz) for 1 h at room temperature. The antibody staining was developed using a diaminobenzidine (DAB) detection system (Catalog # 760124, Ventana Medical Systems, Tucson, AZ, USA) according to the manufacturer's protocol, counterstained with hematoxylin, and examined under a microscope (Nikon E600, Japan).