Anti ho 1

Formalin-fixed, paraffin-embedded lung tissue sections were deparaffinized with xylene, rehydrated gradually with graded alcohol solutions, and then washed with deionized water. After antigen retrieval and blocking, the sections were incubated with rabbit polyclonal anti-HO-1 (1:200, Abcam) and rat monoclonal anti-CD68 (1:100, Bio-Rad) antibodies at 4 °C overnight. The sections were washed in PBS and then incubated with a 1:400 dilution of fluorophore-labeled anti-rabbit (Alexa 555) and anti-rat (Alexa 488) antibodies at room temperature for 2 h. DAPI was used for nuclear counterstaining. The slides were then washed with PBS and mounted with mounting medium containing 4′,6-diaminido-2-phenylindole (Vector Laboratories). Images were acquired with a confocal microscope (Leica TCS SP5 Spectral Confocal Microscope). For each experiment, at least six different fields were examined.

Nuclear and cytoplasmic proteins were extracted with nuclear and cytoplasmic extraction reagents kits (Pierce, Rockford, IL, USA) according to the manufacturer's protocol for Nrf2, Ho1, and Nqo1 detection. Protein concentrations were measured using BCA protein assay. Proteins were separated by 10% or 12% SDS-PAGE and then transferred onto PVDF membranes. The transferred membranes were blocked with TBS-T (10 mmol/L Tris-HC1, 150 mmol/L NaC1, 0.1% Tween-20) containing 5% skim milk for 1 h at room temperature. The membranes were washed in TBS-T (10 min × 3) and then the membranes were incubated overnight at 4°C with anti-Ho1 (Abcam, Cambridge, UK), anti-Nqo1 (Sigma, St. Louis, USA), anti-Nrf2 (Santa Cruz, CA, USA), anti-β-actin (Abmart, Shanghai, China), and anti-PCNA (Santa Cruz, CA, USA) in TBS-T. The membranes were washed three times again in TBS-T; the membranes were incubated with horseradish peroxidase conjugated to anti-rabbit IgG (Weiao Biotech Ltd, Shanghai, China) overnight at 4°C. The expression of targeted proteins was determined using Odyssey machine and optical density of band was analyzed by using Image J software.

GSS was purchased from the HANKOOK SHINYAK Corporation (Chungcheongnam-do, South Korea). Primary antibodies used for Western blotting were as follows: anti-ionized calcium-binding adapter molecule 1 (Iba-1; 1 : 1000; Wako, Japan), anti-glial fibrillary acidic protein (GFAP; 1 : 3000; Millipore, MA, USA), anti-survival motor neuron (SMN; 1 : 1000; Santa Cruz Biotechnology, CA, USA), anti-TLR4 (1 : 1000; Santa Cruz Biotechnology), anti-CD14 (1 : 1000; BD Pharmingen, CA, USA), anti-COX2 (1 : 1000; Abcam, MA, USA), anti-transferrin (1 : 1000; Santa Cruz Biotechnology), anti-HO1 (1 : 1000; Abcam), anti-Bcl2 associated X (Bax, 1 : 1000; Santa Cruz Biotechnology), anti-phospho 5′-adenosine monophosphate-activated protein kinase (pAMPK; 1 : 1000; Cell Signaling, MA, USA), anti-AMPK (1 : 1000; Cell Signaling), and anti-phospho mammalian target of rapamycin (mTOR; 1 : 1000; Cell Signaling). Anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1 : 1000; Santa Cruz Biotechnology) was used to control for protein loading. Peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology.

The TMA was processed and fixed using routinely established protocols and stained as previously described [28 (link)]. Briefly, immunohistochemistry was done using the streptavidin-biotin-peroxidase complex system LSAB + kit, horseradish peroxidase (DAKO). Endogenous peroxide activity was quenched using hydrogen peroxide in distilled water (3%). Antigen retrieval was done by microwaving. Tissue slides were incubated overnight with the following primary antibodies: monoclonal mouse anti-ANXA2 (1:200) from Cell Signaling Tech, (Danvers, MA, USA) and rabbit polyclonal anti–HO-1 (1:50) from Abcam (Burlingame, CA, USA); this was followed by sequential incubations with biotinylated link antibody and peroxidase-labeled streptavidin complex. The peroxidase reaction was conducted, under microscope, using 3,3′-diaminobenzidine. Slides were counter-stained with Mayer’s hematoxylin and analyzed by standard light microscopy. Negative control slides were prepared by substituting primary antiserum with PBS. For semiquantitative analysis, the degree of staining was rated as high, moderate, low, or not detectable (3+, 2+, 1+, and 0, respectively); the staining was also observed for localization.

The human keratinocyte cells (HaCaT) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). HaCaT and HaCaT-ARE (stably transfected with 3xARE-luciferase gene) [23 (link)] cells were maintained at 37 °C in a humidified atmosphere of 5% CO₂/95% air in RPMI1640 supplemented with 10% heat-inactivated FBS and 50 U/mL penicillin/streptomycin mixture (Invitrogen, Carlsbad, CA, USA). MCR was kindly provided by Dr. Heejung Yang of the Kangwon National University. For western blotting, anti-Nrf2 (Cat: ab137550, Abcam, Cambridge, MA, USA), anti-HO-1 (Cat: ab68477, Abcam), anti-Lamin A/C (Cat: sc20681, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and GAPDH (Cat: sc25778, Santa Cruz Biotechnology) were used.

Standards of iAs³⁺ (NaAsO₂), iAs⁵⁺, sodium methyl arsenate, and sodium dimethylarsinate were purchased from Sigma-Aldrich Co. (San Francisco, USA). Commercially available GSPE powder was obtained from Solarbio Science & Technology Co. Ltd. (Beijing, China; purity ≥ 95%). Human hepatocytes (L-02 cells) were purchased from Obio Technology Co. Ltd. (Shanghai, China).
SOD, GSH, MDA, sulfhydryl (-SH), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and ROS kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Anti-Nrf2, anti-HO-1, anti-NQO1, and anti-GST antibodies were obtained from Abcam Ltd. (Abcam, Cambridge, UK). ML385 (Lot no. 846577-71-9), an Nrf2 inhibitor, was purchased from MedChemExpress (New Jersey, USA).
Dulbecco's modified Eagle's medium (DMEM), hyperglycemic medium, fetal bovine serum (FBS), and trypsin were all purchased from Gibco (California, USA).