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RPMI-1640 medium and RIPA buffer were purchased from ThermoFisher Inc. (Carlsbad, CA, USA). Fetal bovine serum (FBS), trypsin, and penicillin-streptomycin were purchased from HyClone (Logan, UT, USA). The primary antibodies were anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antibody (sc-32233; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Glo-1 monoclonal antibody (sc-133214; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-nuclear factor erythroid 2-related factor 2 (Nrf-2) monoclonal antibody (sc-722; Santa Cruz Biotechnology, Santa Cruz, CA, USA). The secondary antibodies were mouse anti-rabbit IgG-HRP (sc-2357; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and goat anti-mouse IgG-HRP (AP124P; Sigma-Aldrich, St. Louis, MO, USA). JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine chloride was purchased from Biotium (Fremont, CA, USA). MG, N-acetyl-cysteine (NAC), and all other chemicals were purchased from Sigma-Aldrich.

Liver tissue samples were obtained from three mice in each experimental group, respectively, after irradiation 48 h. Samples were sectioned (5 µm thickness) and fixed with 4% paraformaldehyde. The sections were stained with H&E and analyzed. IHC staining was performed on the paraffin-embedded sections. After de-paraffinization, the sections were blocked with 5% bovine serum albumin for 1 h at room temperature in PBS containing 0.1% Triton X-100. The sections were then incubated overnight at 4 °C with the polyclonal anti-Nrf2 antibodies (1:500) (sc-722, Santa Cruz Biotechnologies, Santa Cruz, CA, USA) and appropriate secondary antibodies. The sections were then gently rinsed three times with PBS followed by hybridization with the secondary antibody at room temperature in a humid chamber. After immunohistochemical staining and counterstaining with DAPI, the sections were examined under the microscope.

Crizotinib (purity ≥ 98%) and sunitinib (purity ≥ 98%) were obtained from Huateng pharmaceuticals-company (Hunan, China). tBHQ and NAC were purchased from Sigma Aldrich (St. Louis, MO, United States) and Solarbio (Beijing, China), respectively. The primary antibodies used in the present study were as follows: anti-Nrf2 (sc-722, Santa Cruz), anti-cleaved caspase3 (c-caspase3) (af7022, Affinity), anti-Bcl2 (ab692, Abcom), anti-Bax (ab32503, Abcom), anti-PARP (bf0719, Affinity), anti-cleaved PARP (af7023, Affinity), anti-cytochrome c (cytc) (ab13575, Abcom). The anti-β-actin (ac006, ABclonal) and anti-Histone H3 (af0863, Affinity) antibody were used as the control.

Whole hearts of mice were pulverized into powder, and lysed in 100 mM NaCl, 50 mM Tris pH7.5, 20 mM β-glycerophosphate, 20 mM NaF, 1% NP-40, 1 mM PMSF, 5 mM EDTA, 10 mM EGTA, 1 mM Na₃VO₄, 1X protease inhibitor cocktail (cOmplete™ Mini, Roche) and clarified by centrifugation (16,000 g for 20 min at 4°C). Protein concentrations were measured using the Protein Assay Dye Reagent Concentrate (#500-0006, Bio-Rad). Aliquots containing 40–50 μg of protein were denatured for 5 min at 95°C in the presence of 10 mM DTT, separated by SDS-PAGE and transferred to nitrocellulose membrane. Immunoblots were probed with antibodies against NOX4 (1:1000, ab133303, Abcam) or NOX4 (1:500, NB110-58849, Novus Biologicals), ANGPTL2 (1:1000, AF 2084, R&D Systems), phospho (Thr180/Tyr182)-p38 MAPK (1:500, 9211, Cell Signaling), p38 MAPK (1:1000, 8690, Cell Signaling), NRF2 (1:500, sc-722, Santa Cruz), KEAP1 (1:500, sc-15246, Santa Cruz), Catalase (1:1000, #219010, Calbiochem^®, Sigma-Aldrich), p22phox (1:500, sc-20781, Santa Cruz), NOX2 (1:500, ab43801, Abcam) and GAPDH (1:1000, AM4300, Ambion). Chemiluminescence was used to detect protein expression (Western Lightning Plus ECL, Perkin Elmer).

Primary antibodies against P62 (pm045), and LC3 (pm036) were purchased from MBL (Japan). An antibody against KEAP1 (#7705) was obtained from Cell Signaling Technology (Beverly, MA). An antibody against NRF2 (sc-722) was purchased from Santa Cruz. An anti-αSMA antibody (ab7817) was obtained from Abcam. Alexa Fluor 488-conjugated goat anti-rabbit, Alexa Fluor 555-conjugated donkey anti-mouse, HRP-conjugated goat anti rabbit, and HRP–conjugated rabbit anti-mouse antibodies, all primers for gene expression analyses, and Lipofectamine RNAiMAX reagents were purchased from Invitrogen. tBHQ (tertiary butylhydroquinone), anti-β-actin, DMEM (high glucose), and 2× Laemmli sample buffer were purchased from Sigma.

Proteins were extracted from cell pellets lysed and quantified using the Pierce BCA Protein Assay kit (Thermo Scientific, Rockford, IL, EUA). After that, proteins were separated by electrophoresis on an SDS-polyacrylamide gel and transferred to a nitrocellulose membrane (GE Healthcare, Waukesha, WI, USA). Membranes were blocked for 1 h in 5% (w/v) milk powder in PBS and incubated overnight at 4 °C with primary antibody against anti-NRF2 (1:500) (#sc-722 Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-MRP1 (1:1000) (#ab3368 Abcam, Cambridge, UK), anti-caspase 3 cleaved (1:200) (#ab2302 Abcam), and anti-βactin (1:1000) (#4967 Cell Signaling). Then, membranes were incubated with the correspondent secondary antibody and a chemiluminescent HRP substrate (Merck Millipore. Burlington, MA, USA) was used to develop the membranes. Each blot was performed twice. The full and uncropped western blots can be found in Supplemental Material.

Prefrontal cortex was fixed in 10% phosphate-buffered formalin for 2.5 hours. Each tissue block was dehydrated in a series of alcohol solutions of 50%, 70%, 96% and 99% and then in Bioclear. Samples were then paraffin-embedded and cut into 7 μm-thick sections. Sections were de-waxed (Bioclear and alcohol in progressively lower concentrations), rehydrated and processed for haematoxylin-eosin and for anti-Nrf2 immunohistochemical analysis according to manufacturer protocol. Primary antibody anti-Nfr2 (rabbit polyclonal, sc-722, Santa Cruz Biotechnology, CA, USA) was applied for 2 hours at room temperature and diluted 1:200 in PBS. The immunohistochemical reactions was revealed with Rabbit specific HRP/DAB detection IHC kit (ab236469). Peroxidase reaction was developed using diaminobenzidine (DAB) chromogen and nuclei were counterstained with haematoxylin. Lastly, sections were dehydrated, cleared with xylene and mounted in Bio Mount (Bio Optica, Milano, Italy). Negative control was performed by omitting the primary antibody. Samples were then observe by means of LEICA DM 4000 light microscopy (Leica Cambridge Ltd., Cambridge, UK) equipped with a Leica DFC 320 camera (Leica Cambridge Ltd.) for computerized images^{62 (link),63 (link)}.