Chemicals

All the chemicals were purchased from Sigma (St. Louis, MO) unless otherwise stated.

Cell culture

Neuroblastoma SH-SY5Y cells were purchased from ATCC (ATCC CRL-2266) and maintained in DMEM medium supplemented with 10% fetal bovine serum. The cells were cultured in a humidified chamber at 37 °C with 5% CO 2 . Cells were plated the day before treatment so that the density of cell culture could reach ~70% confluence. Hydralazine was diluted in culture medium from a stock solution. The final concentration of hydralazine and the duration of the treatment were indicated in the text and the Figure legends.

Oxidative stress was induced in cells with different concentrations of stressors (e.g., hydrogen peroxide or rotenone) to test the efficacy of hydralazine. Hydrogen peroxide treatment was done in 5% serum containing medium. At the end of the treatment, cells were collected and washed once in ice-cold PBS buffer, followed by lysis with RIPA buffer (1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate, pH 7.2) supplemented with cocktails of proteases and phosphatases inhibitors (Thermo Fisher, Waltham, MA) on ice for 1 h with occasional stirring. The cell lysates were then centrifuged at 10,000 × g for 15 min at 4 °C. Supernatants were collected and protein concentration was measured using the bicinchoninic acid (BCA) assay (Pierce, 23228).

Protein carbonyl assay

Carbonyl content of hydrazine and hydralazine-treated SH-SY5Y cells with or without H 2 O 2 stress were measured using Protein Carbonyl Content Assay Kit (Sigma-Aldrich, MAK094). Interfering nucleic acids were removed using 10% streptozocin solution and carbonyl content of the supernatant was measured spectrophotometrically at 375 nm by adding 2,4-dinatrophenylhydrazine (DNPH) followed by detection of dinitrophenyl hydrazine adduct using a micro plate reader following vendor instructions. The protein content of each sample was determined using the BCA assay.

Cell culture in SILAC media and lysate preparation

SHY-SY5Y cells were grown in Dulbecco’s modified Eagle’s medium containing either unlabeled l-Proline, l-arginine (Arg0) and l-lysine (Lys0) or l-Proline, heavy isotope-labeled l-arginine-13C 6 14N 4 (Arg10) and l-lysine- 13 C 6 - 15 N 2 (Lys8 ) (Cambridge Isotope Laboratories, Inc.) supplemented with 10% dialyzed fetal bovine serum (Thermo Fischer, Waltham, MA). Light labeled cells were left untreated to serve as control and heavy labeled cells were treated with 10 μm of hydralazine for 24 h. After treatment, cells were harvested by trypisinization, washed three times with cold PBS and lysed in a buffer containing 6 M Urea, 2 M Thio-urea, 1% SDS and 100 mm Tris/HCl, pH 8.0 with protease (Thermo Fischer, Waltham, MA) and phosphatase inhibitors. After incubation for 15 min at RT and sonication, the samples were clarified by centrifugation for 15 min at 20,000 × g. Protein content was determined using the 660 nM protein assay kit (Thermo Fisher Scientific, 22660) according to the manufacturer’s instructions.

Protein digestion and peptide fractionation

Equal amounts of protein from control and hydralazine-treated cells were mixed in a 1:1 ratio and digested in solution. The mix digest (300 μg) was then fractionated into six fractions via strong cation exchange (SCX). SCX cartridges were pre-equilibrated with a buffer composed of 0.5% acetic acid and 2% ACN (wash buffer). The digest was then loaded onto the column and washed with wash buffer and subsequently eluted with a buffer containing ammonium acetate (30, 50, 70, 80, 120, and 500 mM), 0.5% acetic acid, and 2% ACN. Eluted peptide fractions were desalted using reverse phase cartridges.

Mass spectrometry for SILAC

All the fractions were analyzed using a Q-Exactive HF mass spectrometer (Thermo Electron, Burlingame, CA) coupled to an Ultimate 3000 RSLCnano HPLC systems (Thermo Electron, Sunnyvale CA). Peptides were loaded onto a 75 µm × 50 cm, 2 µm Easy-Spray column (Thermo Electron, Sunnyvale, CA) and separated using a 120 min linear gradient from 1 to 28% acetonitrile at 250 nl/min. The Easy-Spray column was heated at 55 °C using the integrated heater. Shotgun analyses was performed using a data-dependent top 20 method, with the full-MS scans acquired at 60 K resolution (at m/z 350) and MS/MS scans acquired at 15 K resolution (at m/z 200). The under-fill ratio was set at 0.1%, with a 3 m/z isolation window and fixed first mass of 100 m/z for the MS/MS acquisitions. Charge exclusion was applied to exclude unassigned and charge 1 species, and dynamic exclusion was used with duration of 15 s.

Western blot analysis

Protein expression was determined by western blot analysis. Equal amount of protein from each sample was run in Tris-glycine SDS-PAGE gel, followed by transfer to PVDF membrane. After blocking the membrane with 5% milk for 1 h at room temperature, the membrane was incubated further for 2 h with antibodies specific for target proteins: NRF2 (NBP1-32822, 1/1000 dilution), pNRF2 (S40) (NB100-80012, 1/1000 dilution) from Novus Biologicals (Littleton, CO); KEAP1 (4617, 1/500 dilution), HMOX1 (5061, 1/500 dilution), NQO1 (3187, 1/1000 dilution), HIF1Aα (3716, 1/1000 dilution), HSF1 (4356, 1/1000 dilution), and lamin B1 (12586, 1/1000 dilution) from Cell Signaling (Danvers, MA); GAPDH (sc-47724, 1/5000 dilution) from Santa Cruz (Dallas, TX); β-Actin (MA5-15739-HRP, 1/2000 dilution) from Thermo Fisher (Waltham, MA) and GCLC (ab190685, 1/1000 dilution), GCLM (ab124827, 1/1000 dilution), and TXN (ab26320, 1/1000 dilution) from Abcam (Cambridge, MA). The membrane was subsequently incubated with species-specific HRP-conjugated secondary antibody followed by incubation with chemiluminescence substrate and imaging. The band intensity of each of the target proteins was quantified using either ImageQuant or Image J software (GE Healthcare, Sweden).

NRF2 knockdown by siRNA transduction

NRF2 was knocked down using a human NRF2 specific siRNA in a lentiviral vector (sc-37030-V, Santa Cruz, CA). A scrambled siRNA was used as negative control. In total, 50% confluent cells grown in polybrene-containing complete medium were treated with siRNA lentiviral particles directly added into the medium. After 2 days of transduction stable clones were selected using 0.8 µM puromycin for 2 weeks. Expression of NRF2 was determined by western blot analysis as described earlier in this method and by quantitative real-time reverse PCR (qRT-PCR).

Fluorescence polarization assay

The direct possible inhibitory effect of hydralazine on a NRF2–KEAP1 interaction was measured using KEAP1–NRF2 Inhibitor Screening Assay Kit (BPS Bioscience, 72020), following the vendor instructions. In total, 25 µl of the prepared master mix, including fluorescently labeled NRF2 peptide, was added to each well in a 96-well plate, followed by adding 5 µl of solution containing corresponding concentrations of hydralazine or sulforaphane as positive inhibitor to each well. The reaction was initiated by adding diluted purified KEAP1 protein. The florescence signal was measured using Spectramax Gemini XPS plate reader (Molecular Devices, Sunnyvale, CA) and data analysis was done as suggested by the vendor.

Cell viability assay

Cell growth was analyzed using the MTT cell viability assay. Briefly, at the end of incubation/treatment, MTT reagent was diluted in culture medium and aliquoted into each well. After incubation for 2 h, the medium was aspirated and DMSO was aliquoted into each well to disrupt the cells and dissolve the intracellular MTT dyes. Absorbance was read at 570 nm wavelength in a 96-well plate reader. The absorbance was read at 490 nm in a 96-well plate reader. Primary cortical neuronal cells were cultured in 96 wells plates for three weeks then treated with 0.1 or 1.0 μM of hydralazine or 1 μM of rotenone for 24 h. Cell viability assay was performed using CellTiter-Glo assay (Promega, G7571).

Co-immunoprecipitation (Co-IP) and immunoprecipitation

Cells were lysed in ice-cold 50 mM Tris-HCl buffer with 0.5% Triton X-100 and protease inhibitors, followed by centrifugation at 12,000 × g for 15 min at 4 oC. Protein in the supernatant from each of the samples was incubated with antibody (either specific for NRF2 or KEAP1), at 4 oC overnight, on a rotator with constant stirring. Protein A/G magnetic beads (Thermo Fisher, Waltham, MA) were added into the antibody–antigen mixture followed by incubation for 1 h at 4 oC on a rotator. The tubes were applied to a magnetic stand to collect the beads, followed by washing in lysis buffer for three times. Finally, the bead-bound antibody–antigen mixture was eluted with equal volume of 1× electrophoresis sample buffer. The eluted protein was subjected to western blot analysis as described earlier. NRF2 IP was done using the same antibody used for Co-IP but with a different lysis buffer (150 mm NaCl, 10 mM Tris-HCl (pH 7.4), 1 mm EDTA, 1 mM EGTA (pH 8), 15 Triton X-100, 0.5% NP-40, protease inhibitors). The NRF2-enriched samples were used for WB analysis and mass spectrometry. IP samples were separated on SDS-PAGE and the NRF2 corresponding bands were cut from the gel before in gel digestion and mass spectrometry.

Cell fractionation

Nuclear and cytoplasmic fractions were separated using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, 78833). Briefly, cells were harvested and washed with ice-cold PBS buffer. The cells were resuspended in ice-cold CER-I buffer and incubated on ice for 10 min, followed by addition of CER-II buffer and incubation for one more minute on ice. The lysate was centrifuged at 16,000 × g for 5 min. The supernatant was the cytoplasmic fraction. The pellet obtained was lysed in ice-cold NER buffer to obtain the nuclear protein fraction.

NRF2 transcriptional activity assay

The transcriptional activity of NRF2 was determined using a luciferase-based transcription activation assay. A vector carrying a NRF2 promoter controlled luciferase gene (firefly luciferase) and a vector carrying the control luciferase (Renilla luciferase) from an ARE reporter kit (BPS Bioscience, 60514) was transiently co-transfected into the cells using Lipofectamine reagents (Thermo Fisher, Waltham, MA). After transduction for 24 h, the cells were treated with hydralazine, hydrazine, N-acetyl cysteine (NAC), Tempol, hydralazine + NAC, or hydralazine + Tempol for another 24 h before being subjected to the luciferase assay with the Dual-Glo Luciferase Assay System (Promega, E2920). Briefly, the cells were incubated with firefly luciferase substrate for 10 min prior to measuring luminescence in a 96-well luminescence plate reader. Subsequently, Renilla luciferase was measured after the addition of Dual-Glo Stop & Glo reagent into the wells with a 10-min incubation. The ratio of luminescence from firefly and Renilla was calculated to normalize and compare NRF2 transcriptional activity.

Quantitative real-time PCR

The relative levels of NRF2 and its target genes mRNAs were measured by qRT-PCR. Total RNA was isolated from SHY-SY5 cells using Aurum Total RNA Mini Kit (Bio-Rad, 7326820). cDNA synthesis was done using 1.5 µg of the total RNA following the manufacturer protocol (Maxima First Strand cDNA Synthesis Kit, Thermo Scientific, K1671). RT-PCR reactions were prepared using PowerUp SYBR Green Master mix (Thermo Fisher Scientific, A25742), specific primers (Sigma) of the target genes (Supplementary Table 1) and an equal amount of the diluted cDNAs. Reactions were performed on a C1000 Thermal cycler (Bio-Rad) machine and data were analyzed by Bio-Rad CFX manager 3.1 software using the ΔΔCq method. All data were normalized to the control using actin as internal control.

Measurement of reactive oxygen species

Superoxide concentration was measured by dihydroethidium (DHE). Black clear bottom 96 well plates were seeded with about 5000 HEK293 tau aggregate-negative (control) or aggregate-positive model cells per well and about 20,000 SH-SY5Y cells. Cells were treated with hydralazine and the superoxide level was measured by incubating cells with DHE (1 µM) for 30 min. Fluorescence was measured by Spectramax Gemini XPS plate reader (Molecular Devices, Sunnyvale, CA) at 370 nm excitation and 420 nm emission wavelengths. Superoxide concentration in worms was measured after hydralazine treatment for 3 days followed by incubation with DHE (6 µM final concentration) for 30 min. Equal number of animals were transferred to a black bottom 96 well plate and fluorescence was measured as above.

Hydroxyl, peroxyl and other reactive oxygen species (ROS) were measured in SH-SY5Y cells using the Cellular Reactive Oxygen Species Detection Assay Kit (abcam, ab113851) according to the vendor protocol. Tert-butyl hydrogen peroxide (TBHP) was used as positive control to generate ROS.

Protein profiling using label-free quantitation

Worms were lysed in 8 M urea, 50 mM Tris-HCl pH 8.0 and 1x protease inhibitor cocktail EDTA-free (Thermo Fisher, Waltham MA) buffer with the aid of sonication. Lysates were centrifuged at 14,000 × g for 15 min at 4 °C and proteins in the supernatant were precipitated using cold acetone. Disulfide bonds were reduced and alkylated (by DTT and IAA respectively) before diluting the solution to 1.8 M with 25 mM Tris-HCl pH 8.0. Proteins were digested overnight at 37 °C with trypsin (Promega, Fitchburg WI) in the presence of 1 mM CaCl 2 . Peptides were acidified with TFA and purified using Oasis HLB plates (Waters, UK). A Dionex Ultimate 3000 UHPLC (Thermo Electron, Sunnyvale CA) was coupled to an Orbitrap Fusion Lumos mass spectrometer for the separation and analysis of tryptic peptides. An Easy-Spray column with 75 µM inner diameter and 50 cm long packed with 2 µM C18 material was used for peptide separation. 0.1% formic acid and 2% (v/v) acetonitrile in LCMS grade water was used as buffer A and 10% (v/v) TFA plus 80% (v/v) acetonitrile in LCMS grade water was used as buffer B. In total, 5 μl of sample were injected and separated using a gradient from 0 to 28% mobile phase B over 180 min (240 min total run time). Source voltage was set to 2.2 kV and capillary temperature to 275 °C in the positive ion mode. Ions within the m/z range of 400–1600 were scanned at the resolution of 120,000. Collision induced dissociation method was used to fragment top 10 MS spectra with 2–7 charge states. Label-free quantitation was also performed to detect and quantify NRF2 protein in the treated SH-SY5Y cells. Samples were prepared as described in the IP section. Gel bands were cut in 1 mm cubes and destained using 400 µl of 50 mM TEAB in acetonitrile (1:1) for 30 min at 37 °C. Destained gel pieces were washed with 100% acetonitrile before being reduced and alkylated (by DTT and iodoacetamide respectively). Proteins in the gel pieces were digested overnight at 37 °C with trypsin (Promega, Fitchburg WI). Peptides were extracted from gel pieces using peptide extraction buffer (66.7% ACN; 5% TFA in dH 2 O) for 15 min at 37 °C and the extract was transferred to a fresh LoBind tube. Gels were washed with 50 µl of 100% acetonitrile and the wash was pooled with the previously collected extracts. Samples were dried down in a vacuum concentrator before clean up using Oasis HLB plates (Waters, UK). MS conditions were the same as above, except the gradient time (120 min instead of 180 min).

MS data processing and Ingenuity pathway analysis

LC–MS/MS raw data files were processed using the latest available MaxQuant software (v.1.5.3.30). Proteins were identified by the Andromeda search engine within the MaxQuant program and the search was performed against the UniProt/Swiss-Prot C. elegans database. We used one multiplicity as standard label free search. Carbamidomethyl cysteine was set as a fixed modification and methionine Oxidation (M) and Acetyl (protein N-term) were used as variable modifications. The protein and peptide false discovery rates and peptide-to-spectrum match (PSM) false discovery rate (FDR) were set to 1%. Match between runs was performed by using a match time window of 0.7 (minimum) and alignment time window of 20 (minimum). The decoy proteins, known contaminants (after quality control using cluster analysis), proteins identified with a single modified peptide and low confidence proteins identified by only one peptide were filtered out. The p-values for all the statistical analysis were calculated using a two-tailed Student's t test as used for normally distributed data (we pre-processed intensities by binary logarithm) unless otherwise stated. Our entire identified proteins with their fold change values was submitted to IPA and the pathway analysis was performed by the Ingenuity Knowledge Base (genes only) as the reference set with direct and indirect relationships included.

C. elegans strains and maintenance

Animals were grown and maintained using standard C. elegans conditions at 20 °C on NGM plates and were fed E. coli strain HB101. N2 worms were used as wild-type and the following mutants and transgenic strains were used from Caenorhabditis Genetics Center (CGC, University of Minnesota): EU1 skn-1(zu67) (IV)/nT1[unc-?(n754);let-?] (IV;V), EU31 skn-1(zu135) (IV)/ nT1[unc-?(n754);let-?] (IV;V), CL2166 dvIs19 [gst-4p::GFP::NLS] (III), CL691 dvIs19 [gst-4p::GFP::NLS] (III); skn-1(zu67)/nT1 [unc-?(n754) let-?] (IV;V), LG333 skn-1(zu135) (IV)/nT1[qIs51] (IV;V); ldIs7 [skn-1b/c::GFP], LG348 skn-1(zu135) (IV)/nT1[qIs51] (IV;V);geIs9 [gpa-4p::skn-1b::GFP+rol-6(su1006)], LG357 skn-1(zu135) (IV)/nT1[qIs51] (IV;V);geIs10 [ges-1p(long)::skn-1c::GFP+rol-6(su1006)], and LD1 ldIs7 [skn-1b/c::GFP+rol-6(su1006)], DA1116 eat-2(ad1116), BR5270 byIs161[Prab-3::F3DK280;Pmyo-2::mCherry], BR6516 byIs194;[Prab-3::F3DK280(I277P)(I308P);Pmyo-2::mCherry], CF1038 daf-16(mu86) (I), SJ4005 zcIs4 [hsp-4::GFP] (V).

Rotenone stress test

Hydralazine, hydrazine, and NaCl were dissolved in water, while curcumin, sulforaphane and rotenone were dissolved in DMSO. Synchronized L1 larvae were placed on NGM plates preloaded with hydralazine, hydrazine, curcumin, sulforaphane or NaCl. After 3 days young adult worms were transferred to fresh NGM plates either preloaded with rotenone alone or rotenone plus any of the abovementioned compounds. Every worm was subjected to a prodding test with a worm pick every day. A worm was scored as dead when not responding to three repeated proddings. Survival curve was plotted using Prism 7.

Lifespan analysis

All lifespan assays were performed at 20 °C using HB101 as food source according to standard protocols26. Worms were synchronized by hypochlorite solution. The L1 worms hatched overnight were transferred to agar plates (Corning Inc.). Hydralazine was added freshly from a 5 mM stock to the NGM media. Water was used as control. The L4 population of worms were randomly split to control or treatment groups in a density of about 20–30 worms per 6 cm plate dish. The first day of adulthood was considered day one. In all the experiments with late-onset administration of hydralazine, synchronized L4 animals were moved to the media supplemented with 5′-fluorodeoxyuridine (FUDR, Cayman chemicals) at a final concentration of 40 µM for 2 days to prevent reproduction, then were moved to FUDR-free NGM plates until the final treatments at day 10. The animals that crawled off the plate, ruptured, or died from internal hatching were censored. Worms were transferred to fresh plates every day after reaching adulthood, and every 2 days after reaching 10 days of age. Prodding test as described above was used to count the number of dead worms. Survival curve was plotted using Prism 7 and the significance of the curves calculated by Log-rank (Mantel-Cox) test. To generate inactive bacteria, the HB101 bacteria, grown overnight, were centrifuged, re-suspended in M9, heated 30 min at 65 °C and kept at −20 °C. The bacteria were added (100 µl) freshly to the plates containing hydralazine or water, as control, before transferring animals57.

RNA interference

Synchronized L1 larvae were placed on NGM plates containing 1 mM IPTG and fed HT115 bacterial strain containing scrambled or skn-1 RNAi plasmids (Julie Ahringer RNAi library clone for skn-1: 1568 bp insert, Chr IV 5,652,318–5,653,885, the sequence targeted includes exons 4, 5, and 6 of skn-1a, and therefore should target all SKN-1 isoforms). All experiments were done at 20 °C.

Fluorescence microscopic imaging

To measure GFP intensity, synchronized populations of worms were anesthetized and arranged on an agarose pad. The intestinal SKN-1::GFP was assayed by confocal microscopy (X40) (Nikon A1R, Nikon Instruments Inc., Melville, NY, USA) (Fig. 4a). The quantification of intestinal SKN-1::GFP was recorded as high (≥15 GFP-positive intestinal nuclei), medium (5–15 GFP-positive intestinal nuclei), or low (≤5 GFP-positive intestinal nuclei) (Fig. 4b). ASI SKN-1::GFP was assayed by a Zeiss AxioImager M2 microscope equipped with a Hamamatsu Flash 4.0 Scientific c-mos camera and Zen2 software (×40) (Fig. 4c). The quantification of ASI SKN-1::GFP was performed with ImageJ using a sliding paraboloid algorithm for reducing the background followed by edge detection. The gst-4p::GFP intensity was measured same as ASI SKN-1::GFP but with ×5 magnification and the quantification was done by ImageJ using the whole worm signal.

Locomotion assays

To measure locomotion, worms were subjected to 30 s video recording on a Zeiss Axio Zoom.V16 fluorescence dissecting microscope equipped with Axiocam 503 and ZEN2 software. Bending rate (the number of body-bends-per-second) was measured by placing live animals on a plate containing M9 buffer, filming for 30 s and counting the number of bends. Healthiness of the worms was measured by the bending rate of young, middle age and old animals. For rotenone experiments synchronized N2 or skn-1(zu135) L1 worms were placed on NGM plates with 100 µM hydralazine for 3 days. Worms were then transferred to new NGM plates containing either 50 µM rotenone plus 100 µM hydralazine, or 50 µM rotenone for 6 h. The results were obtained from three individual trials.

Pumping rate assay

Synchronized day 4 adult wild-type worms were used to count the number of contractions in the pharyngeal terminal bulb as described by Wilkinson et al.26. Total number of pumps/min was counted using a hand-held counter under a dissection scope for at least 10 worms on the bacterial lawn and three independent trials were carried out. Worms were treated with hydralazine for 24 h before doing the assay. Mutant eat-2 animals were used as negative control.

Bacterial growth rate

To study the possible growth inhibitory and anti-proliferative effect of hydralazine on HB101 bacteria, they were grown in 96-well plates using liquid LB in the presence of different concentrations of hydralazine. In total, 1–500 dilution cultures of HB101 were grown overnight with shaking at 37 °C. Absorbance (OD 595 nm) was measured every 1 h using a microplate reader.

Lipofuscin measurement

Autofluorescent lipofuscin intensity was measured as described58. Synchronized populations of young adult and middle age animals were treated with hydralazine for 3 and 10 days, respectively, transferred to 96-well black plates (30 worms per well) and the signal was measured using a Spectramax Gemini XPS plate reader (Molecular Devices, Sunnyvale, CA) at 340 nm excitation and 430 nm emission wavelengths.

Fecundity assay

Synchronized L4 animals were transferred to new NGM plates (10 worms per plate) containing H 2 O as vehicle or hydralazine as treatment. Animals were moved to new plates every day until the end of the reproductive period and the number of progeny in the original plates were counted after 24 h to allow all the fertile eggs to hatch.

Data availability

Proteomics data have been deposited in Proteomexchange under accession code PXD005618.