Hydroethidine

Antibodies used were anti-NRF2 (Abcam Cambridge, UK), anti-phospho-histone H₂A.X (Ser139), anti-phospho-ATM (Ser1981) (Merck KGaA, Darmstadt, Germany), anti phospho-ERK 1/2 (Cell Signaling Technology, Danvers, MA, USA), and anti-heme oxygenase 1 (HO-1) (Proteintech, Rosemont, IL, USA). ThermoFisher Scientific provided the Alexa Fluor-488 conjugated Goat Anti Rabbit and Alexa Fluor-555 conjugated Goat Anti Mouse secondary antibodies. The MEK 1/2 chemical inhibitor U0126 was purchased from Promega (Beijing, China). The ATM chemical inhibitor ku55933 was from Santa Cruz (Dallas, TX, USA). The chemical inducer of NRF2 tert-butylhydroquinone (tBHQ) was purchased from Sigma (Merck KGaA, Darmstadt, Germany). Hoechst 33342 and hydroethidine were purchased from ThermoFisher Scientific.

Superoxide anions levels were assessed in situ using the oxidative fluorescent dye, hydroethidine (Cat# D11347, 1:5000; Thermo Fisher Scientific), as described previously.5 Ethidium bromide fluorescence was quantified using an image analyzer system (Image J 1.34; NIH).

ROS production was evaluated by culturing PANC1 and ASPC1 spheroids in Hydroethidine (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s protocol. Stained samples were evaluated by flow cytometry (FACS Canto, BD, Franklin Lakes, NJ, USA) and results analyzed with BD FlowJo software. The production of superoxide by mitochondria was evaluated by the fluorescent MitoSOX™ Red reagent (Thermo Fisher Scientific, Waltham, MA; USA), according to the manufacturer’s protocol and by microscopy. Briefly, PANC1 and ASPC1 spheroids were dissociated and plated on cover slips. Subsequently, the cells were treated as reported in figure legend with domatinostat (0.5 μM). Then, the media was removed and pre-warmed (37 °C) staining solution containing MitoSox probe was added for 15 min at 37 °C. The staining was photographed by Opera Phenix microscope (PerkinHelmer,Waltham, MA USA) and the positive cells are counted by Harmony software (PerkinHelmer,Waltham, MA, USA).

The treated cells (5 x 10⁴ cells/mL) were collected by centrifugation (450 x g/5 minutes at 4°C). The supernatant was discarded and pellet was resuspended with 1 mL fresh prewarmed FBS-free MEM with addition of 1 μL of 50 μM tetramethylrhodamine ethyl ester (TMRE; Thermo Fisher Scientific, Waltham, MA, USA), 5 mM nonyl acridine orange (NAO; Sigma-Aldrich, St. Louis, MO, USA), 10 mM hydroethidine (HE; Thermo Fisher Scientific, Waltham, MA, USA) or 10 mM 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA; Thermo Fisher Scientific, Waltham, MA, USA). The cells stained with TMRE or NAO were incubated for 15 minutes at 37°C whereas cells stained with HE and DCFH-DA were incubated for 30 minutes at 37°C in the dark. After incubation, the cells were centrifuged (450 x g/5 minutes at 4°C) and pellet was washed with 1 mL chilled PBS solution. The supernatant was discarded and 500 μL of chilled PBS was used to resuspend the pellets. The stained cell suspension was transferred to flow tubes and analyzed using FACSCanto II Flow Cytometer (BD Biosciences, San Jose, CA, USA). [dx.doi.org/10.17504/protocols.io.bdqii5ue; dx.doi.org/10.17504/protocols.io.bdqji5un; dx.doi.org/10.17504/protocols.io.bdqki5uw; dx.doi.org/10.17504/protocols.io.bdqmi5u6]

For measurement of intracellular Superoxide, primary hepatocytes were stained with 5 μmol/L hydroethidine (superoxide indicator) (Thermo Fisher Scientific, USA). Stained cells were analyzed with NovoCyte Quanteon flow cytometer (Agilent Technologies, Inc), and acquired data were analyzed with NovoExpress software (Agilent Technologies, Inc) and FlowJo software (TreeStar, Ashland, OR).

The following reagents and kits were used in the present study: Rabbit polyclonal anti-tyrosine hydroxylase (TH; cat. no. 2792; Cell Signaling Technology, Inc., Boston, MA, USA), rabbit polyclonal anti-OX-42 (cat. no. orb11009; Biorbyt, Cambridge, UK), hydroethidine (Molecular Probes, Eugene, OR, USA), LPS (Sigma-Aldrich, St. Louis, MO, USA), heparin (Qianhong Bio-pharma Co., Ltd., Changzhou, China), SST (ProSpec, East Brunswick, NJ, USA), biotinylated goat anti-rabbit secondary antibody (cat. no. A0277) and horseradish peroxidase (HRP)-labeled streptavidin (Beyotime, Shanghai, China), bicinchoninic acid (BCA) kit (Beyotime), Rat TNF-α ELISA kit, Rat IL-1β ELISA kit and Prostaglandin E2 ELISA kit (PGE2; USCN, Wuhan, Hubei, China).

Three days after final MPTP injection, hydroethidine (Molecular Probes; 1 mg/mL in PBS containing 1% dimethyl sulfoxide) was administered intraperitoneally. After 15 min, the animals were transcardially perfused and postfixed, and the brains were cut into 30 μm sections. hydroethidine histochemistry was performed for the in situ visualization of O₂⁻- and O₂⁻-derived oxidants as previously described [23 (link)]. The oxidized hydroethidine product, ethidium, was examined by confocal microscopy (Olympus).