Anti lc3 1 and 2

Anti-CD31, Anti-α-SMA, Anti-IL-6 and Anti-VE-cadherin were purchased from Abcam (ab16669, ab7817, ab233706, ab33168, Abcam; Cambridge, UK). Autophagy related antibodies (Anti-Beclin1, Anti-LC3 I and II, Anti-P63, Anti-SQSTM1) and Anti-GAPDH antibodies were purchased from Cell Signaling Technology (4445,2524,8242, CST; Boston, USA). Anti-fibronectin antibodies were brought from Becton Dickinson and Company (610077, BD; New Jersey, USA). Anti-IL6 monoclonal IgG, control monoclonal IgG and Human recombinant IL-6 were purchased from Merck (I7901, I8765, HY-P7044, Merck; Darmstadt, Germany).3-MA and curcumin were purchased from Selleck chem (S2767, S1848, Selleck; Houston, USA). Myeloperoxidase (MPO) Activity Assay Kit was purchased from SIGMA (mak068, SIGMA; Saint Louis, USA). Superoxide dismutase (SOD) Activity Assay Kit was purchased from SIGMA (19160, SIGMA; Saint Louis, USA).

The hearts were fixed in 4% paraformaldehyde in PBS and embedded in paraffin wax. Tissue sections (5 μm thick) were cut and stained with hematoxylin and eosin (H&E) to determine the level of inflammation. To evaluate the tissue-specific expression of RIP1, RIP3, MLKL, and LC3 I and II, we performed immunohistochemistry. In brief, control and the respective experimental group of tissue sections were incubated with mouse monoclonal anti-RIP1 antibodies (abcam, ab72139), rabbit polyclonal anti-RIP3 antibodies (abcam, ab62344), rabbit polyclonal anti-MLKL (Affinity Bioscience, DF7412), or rabbit polyclonal anti-LC3 I and II (Cell Signaling Technology, 12741S). The specificity of these antibodies was confirmed by Western blots (data not shown). The degree of myocardial infiltration and fibrosis was determined by two independent investigators in a double-blinded manner, and histology was scored as follows: 0, no infiltration; 1, <25%; 2, 25 to 50%; 3, 51 to 75%; 4, >75%.