3 nitrotyrosine 3 nt

Liver tissues fixed in 4% paraformaldehyde were embedded in paraffin using standard procedures. Liver tissue sections were prepared and immunohistochemical staining was performed according to the manufacturer’s instructions. Liver tissue Section 4 µm in thickness were stained with hematoxylin and eosin (H&E) or immunohistochemical characterization was performed using antibody specific to 8-oxo-2′-deoxyguanosine (8-oxo-dG; Trevigen, Inc., Gaithersburg, MD, USA), anti-4HNE (JalCA, Tokyo, Japan), 3-nitrotyrosine (3NT; Millipore, Darmstadt, Germany), and F4/80 (Abcam). Liver sections were stained with Schiff’s reagent (Sigma-Aldrich, St. Louis, MO, USA) for 20 min, followed by counterstaining with hematoxylin solution for 2 min. Sirius Red staining was performed to reveal collagen deposition. The slides were deparaffinized, hydrated with xylene and ethanol, and then incubated with picric acid solution containing 0.1% Fast-green FCF and 0.1% Direct Red 80 for 2 h. All steps were performed at room temperature and the tissues were rinsed with tap water after each step. The stained sections were photographed using a microscope equipped with AxioCam software (Carl Zeiss, Jena, Germany).

Briefly, heart tissues were homogenized with RIPA lysis buffer (Santa Cruz Biotechnology, CA). Total proteins were extracted and separated on SDS-PAGE gels, and then proteins were transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA). The membrane was blocked with 5% nonfat dried milk for 1 h and then incubated overnight at 4°C with the following primary antibodies: intercellular adhesion molecule 1 (ICAM-1, Santa Cruz Biotechnology, CA), tumor necrosis factor alpha (TNF-α, Abcam, Cambridge, MA), plasminogen activator inhibitor-1 (PAI-1, BD Bioscience, Sparks, MD), 3-nitrotyrosine (3-NT, Millipore, Billerica, MA), 4-hydroxy-2-nonenal (4-HNE, Alpha Diagnostic International, San Antonio, TX), B-cell lymphoma 2 (BCL-2) and BCL2-associated X protein (BAX) (Cell Signaling, MA), cleaved-caspase-8 (Cell Signaling, MA), apoptosis inducing factor (AIF) (Cell Signaling, MA), caspase-12 (Exalpha Biologicals, MA), C/EBP homologous protein (CHOP; Santa Cruz Biotechnology, CA) GAPDH (Santa Cruz Biotechnology, CA), and cleaved-caspase-3 (Cell Signaling, MA). After three washes with Tris-buffered saline (pH 7.2) containing 0.1% Tween 20 (TBST), membranes were incubated with appropriate secondary antibodies for 1 h at room temperature. Antigen-antibody complexes were then visualized using an enhanced chemiluminescence kit (Thermo Scientific, Rockford, IL) [22 (link)].

For our sample, we purchased doxorubicin (D1515) and kirenol (52659-56-0) from Sigma-Aldrich Co. (St. Louis, MO, USA). A terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining kit (Roche, cat. no. 11684817910), antibodies against pAKT (# 44-621G), AKT (# 44-609G), pPI3K (#PA5-104853), PI3K (# PA5-29220), BCL2 (# PA5-27094), PARP (PA5-16452), HO-1 (# PA5-77833), NQO1 (# PA5-82294), pNrf2 (# PA5-105664), β-actin (# PA5-78716), and Lamin B1 (# PA5-19468), ANP (# 711569), MMP9 (PA5-13199), MMP2 (PA5-85197), and rhodamine phalloidin (R415) were sourced from Invitrogen, Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Furthermore, we purchased anti-cleaved caspase-3 (ab32042) and Anti-BNP antibody (ab19645) from Abcam (Branford, CT, USA) and Lipofectamine 2000 (11668027) and Nrf2 siRNA from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). We also procured 3-nitrotyrosine (3-NT, 1:3000 dilutions, Millipore, Billerica, MA), 4-hydroxy-2-nonenal (4-HNE, Alpha Diagnostic International, San Antonio, TX, USA), and plasminogen activator inhibitor-1 (PAI-1, BD Biosciences, San Jose, CA, USA).

Western blotting was performed as described previously [30 (link), 35 (link)], and MT expression was detected using a modified western blotting protocol [24 (link)]. Primary antibodies against the following were used: 3-nitrotyrosine (3-NT; 1 : 1,000; Millipore, Billerica, MA, USA); Nrf2 (1 : 1,000; Abcam, Cambridge, MA, USA); α-smooth muscle actin (α-SMA; 1 : 1,000; Abcam); connective tissue growth factor (CTGF; 1 : 1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA); fibronectin (1 : 1,000; Abcam); tumor necrosis factor α (TNF-α; 1 : 1,000; Abcam); NACHT, LRR, and PYD domain-containing protein 3 (NLRP3; 1 : 1,000; Abcam); NAD(P)H:quinone oxidoreductase 1 (NQO1; 1 : 1,000; Santa Cruz Biotechnology); superoxide dismutase-2 (SOD-2; 1 : 5,000; Santa Cruz Biotechnology); catalase (CAT; 1 : 5,000; Santa Cruz Biotechnology); β-actin (1 : 8,000; Santa Cruz Biotechnology); and MT (1 : 1,000; DakoCytomation, Carpinteria, CA, USA).
The protein content was determined by measuring the gray values of bands using Image Lab (Bio-Rad).