Lc3b 2

For immunohistochemistry or immunofluorescent staining, paraffin-embedded heart sections were deparaffinized, rehydrated in xylene and graded ethanol, treated with antigen retrieval solution in boiled water for 20 min, and subjected to hydrogen peroxide for 30 min after cooling down. Tissue sections were blocked with 5% BSA in room temperature for 1 h prior to incubation overnight at 4°C with primary antibodies against cleaved caspase-3 (Cell Signaling Technology 9661) and LC3 BII (Cell Signaling Technology 12741). After incubation with secondary antibody, for immunohistochemical analysis, tissue sections were stained with diaminobenzidine (DAB) (Servicebio, Wuhan, China) and counterstained with hematoxylin, followed by light microscope observation (Olympus); for immunofluorescent analysis, tissue section images were captured by a fluorescent microscope (Olympus). The positive staining areas were calculated and analyzed by Image-Pro Plus 6.0 software.

MDA-MB-231 shC and PHD2 knockdown #3 cells (2×10⁵ cells per plate) were plated on 6 cm dishes and cultured for 1 day in full serum. 16 h before treatment, the cells were cultured in starvation medium (DMEM containing 0.1% FBS), supplemented with either cycloheximide (10 mM), or cycloheximide (10 mM) together with the lysosome inhibitor chloroquine (100 mM) for 16 h. To inhibit the proteasome, the cells were starved and pre-treated with cycloheximide for 16 h, followed by 2 h pre-treatment with MG132 (10 mM). Afterwards the cells were treated with EGF (100 ng/ml) for 0, 1, 2, 3 and 4 h, cell lysates were prepared as described above. To verify lysosomal and proteasomal inhibition, lysates from treated cells were checked for accumulation of LC3B II (#2775, Cell Signaling) and ubiquitin (sc-9133, Santa Cruz).

The homogenized ventricles tissue or cultured cells were incubated in lysis buffer (pH 7.4) containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 25 mM NaF, 1% Triton-X 100, 1% NP-40, 0.1 mM Na₃VO₄, 12.5 mM b-glycerophosphate, 1 mM phenylmethanesulfonylfluoride (PMSF) and complete protease inhibitor cocktail. Insoluble heart tissues or cell fractions were removed by centrifugation at 13,000 × g, 4°C for 30 min. The concentration of the extracted proteins was measured by bicinchonininc acid assay (Sigma-Aldrich, St. Louis, MO, USA). The extracted proteins were boiled for 3–5 min. in 5× loading buffer. The expression of AT1 receptor, LC3b-II, beclin-1 and phosphorylation of Akt (p-Akt) were detected by immune-blotting with antibodies against AT1 receptor (catalogue no.sc-1173G; Santa Cruz Biotechnology, Santa Cruz, CA, USA), LC3b-II (catalogue no. 2775; Cell Signaling Technology, Danvers, MA, USA), beclin-1 (catalogue no. 3495; Cell Signaling Technology), and p-Akt (catalogue no. 9275; Cell Signaling Technology), and detection was performed by using an ECL Western-blotting Detection Reagents (RPN2106; GE Healthcare, Little Chalfont, Buckinghamshire, UK) visualized densitometry with LAS-300 Image software.