Supplementary Data
Title: Morin mitigates acetaminophen-induced liver injury by potentiating Nrf2 regulated survival mechanism through molecular intervention in PHLPP2-Akt-Gsk3β axis
1
Fatima Rizvi
1,2
, Alpana Mathur
2
, Poonam Kakkar
1,2
Supporting Materials and Methods
Materials and Reagents
Antibodies and chemicals including ERK1/2 (4695), phospho-ERK1/2 (9101), SAPK/JNK
(9252), phospho-SAPK/JNK (4668), phospho-Akt Ser473 (4060), phospho-Akt Thr308 (2965), phospho-GSK3β Ser9 (9323), phospho-PDK1 Ser241 (3438), phospho-PTEN Ser380 (9551),
Cox-IV (11967), anti-rabbit Alexafluor 555 conjugate (4413) were purchased from Cell
Signaling Technology (Danvers, MA). Antibodies against caspase-12 (ab62484), HMGB1
(ab79823) and NALP3 (ab17267) were obtained from Abcam (Cambridge, UK).
FBS, 100X antimycotic and antibiotic solution, Collagenase (type IV), OPTI-MEM reduced serum medium, William’s medium E, Lipofectamine RNAiMAX Transfection Reagent,
CellTracker™ Green CMFDA dye and antibody against caspase-1 (AHZ0082) were procured from Invitrogen (Carlsbad, CA). Silencer Select Pre-designed siRNA against Nfe2l2
(ID:s136127), negative control unlabelled siRNA were supplied by Ambion (Austin, TX).

Chemicals and antibodies like Nrf2 (sc-30915), Keap1 (sc-33569), PARP1 (sc-7150), β-actin
2
(sc-81178), Gapdh (sc-25778), Ubiquitin (sc-166553), Lamin b (sc-6216), HO1 (sc-10789),
NQO1 (sc-16463), Akt1 (sc-5298), Fyn kinase (sc-16-G), caspase-3 (sc-7148), caspase-9 (sc-
8355), Bax (sc-493), Bcl2 (sc-492-G), AIF (sc-9416), EndoG (sc-26923), cytochrome c (sc-
8385), p-Akt1/2/3 Ser 473 (sc-101629), agarose conjugated phospho-Thr antibody (sc-5267AC), secondary antibodies, normal goat sera, normal rabbit sera and Protein A/G PLUS Sepharose beads were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
CNBr-activated Sepharose beads were purchased from GE-Healthcare (GE Healthcare
BioSciences Ltd., Kowloon, Hongkong).
All other reagents or chemicals including acetaminophen, morin hydrate, TOX7 (In Vitro
Toxicology Assay) Kit, tBHP, pNPP substrate, PHLPP2 antibody (SAB1300919), antinitrotyrosine antibody (N0409), anti-goat FITC conjugated secondary antibody (F7367), Hoechst
33258 and dexamethasone were procured from Sigma (St Louis, MO, USA) unless otherwise mentioned.
Serum analysis
Activities of AST, ALT and ALP were measured using clinical auto-analyser (RANDOX
Laboratories Ltd., UK). Quantitative determination of serum albumin was done using colorimetry-based commercial diagnostic kit (Spinreact, Ctra Santa Coloma, Spain). LDH leakage into serum was assessed using TOX7 kit. To analyze HMGB1 levels, serum proteins were quantified and 50µg samples were run across 10%SDS-PAGE gels and transferred onto

PVDF membrane. To ensure equal loading, the membrane was stained with Ponceau Red
(Sigma). After incubation with anti-HMGB1 antibody and secondary anti-rabbit antibody, the
3 bands were visualized using Immobilon western chemiluminescent horseradish peroxidase substrate kit (Millipore Corporation, Billerica, MA) on ImageQuant LAS 500 detection system
(GE Healthcare, Upsala, Sweden).
Histology and immunohistochemistry
Formalin-fixed liver samples were embedded in paraffin. Liver tissues were cut to obtain 5μm sections using Leica RM2155 rotary microtome (Leica Microsystems, Nussloch, Germany) and stained with hematoxylin and eosin (H&E) for evaluation of the areas of liver damage. Slides were subjected to histopathological examination using Nikon ECLIPSE 80 i upright microscope
(Nikon Corporation, Japan) microscope attached with digital camera.
For immunohistochemistry analysis, 5μm liver sections were adhered to slides coated with Meyer’s albumin and incubated at 60°C for 2h for drying. Following deparaffinization using standard protocols, the slides were incubated at 60°C for 24h in antigen retrieval solution containing 10mM Tris (pH 9.0), 1mM EDTA and 0.05% Tween 20. Slides were thereafter washed with 0.03% TBST and blocked in 1% BSA in TBS for 2h at room temperature. Primary antibody was applied at a dilution of 1:100 and incubated overnight at 4°C. Slides were rinsed thrice with gentle agitation in 0.03% TBST followed by application of labeled secondary antibody at a dilution of 1:200. To counterstain nuclei, slides were incubated in either 2µM
Hoechst 33258 (to stain nuclei blue) or 0.25µg/ml propidium iodide (to stain nuclei red) for 15 min. Additionally, while counterstaining with PI, cytoplasmic staining was avoided by treating the slides with 20µg/ml RNase solution for 30 min prior to PI staining. The sections were viewed

under Leica DMLB Fluorescence Microscope (Wetzlar, Germany) at 10X objective magnification.
Giemsa Staining
Differential staining for leukocytes was performed by Giemsa stain (Sheehan and Hrapchak,
1980) on 5-μm thin sliced liver sections that were deparaffinized in xylene and were dehydrated in increasing concentrations of isopropyl alcohol. Sections were then dipped in methanol for
2min and stained with Wright Stain for 5min and the excess stain was washed off with distilled water. Following this, slides were kept in Sheehan’s Giemsa solution for 45min at room
4 temperature. After this, sections were dehydrated sequentially in acetic acid (1min), distilled water (1min), 95% alcohol (1min), 100% alcohol (2min) and xylene (3min) and were then coverslipped using DPX as mountant.
Lipid peroxidation assay
Oxidative stress mediated lipid peroxidation was estimated using method by Niknahad et al.,
(2003) with slight modifications. To the 50μl of fresh liver tissue lysates (diluted with distilled water), 25μl of 40%TCA and 100ul of 1.3% thiobarbituric acid were added. The samples were kept for 20 min in boiling water bath and then the reaction was stopped by placing the samples on ice. The samples were then centrifuged at 2600rpm for 10min to collect supernatant.
Absorbance was recorded at 530nm and 630nm using SpectraMax Plus384 Microplate Reader
(Molecular devices, CA, USA). Lipid peroxidation was calculated using 1,1’,3,3’ tetramethoxy propane as standard.

Estimation of protein carbonyl content
Protein Carbonyl content was determined in fresh liver tissue lysate using method by Dalle-
Donne et al., (2003). Briefly, 50μl lysate was added to 950μl of PBS (phosphate buffered saline) and was centrifuged twice for 10 min at14000 rpm to eliminate the particulate matter. The
5 diluted proteins were then concentrated by precipitation with 50%TCA (20% final concentration) and the proteins were collected as pellet after centrifugation for 3-5 min. To the protein pellets,
10mM DNPH (in 2N HCl) was added, while, only 2N HCl was added to the corresponding blanks of each sample. The pellet was loosened by regular vortexing every 10min and incubated in dark for 1h at room temperature. Proteins were then re-precipitated with TCA (20% final concentration), pelleted and washed thrice with ethanol/ethyl acetate (1:1, v/v) to remove excess
DNPH. These samples were then suspended in 6M guanidinium hydrochloride (GdmCl, dissolved in 2N HCl) and were regularly vortexed. Carbonyl contents were determined from the absorbance at 366nm and using molar extinction coefficient of 22000 M-1cm-1.
Estimation of GSH and total thiol content
GSH was estimated using o -phthalaldehyde (OPT) as previously established (Hissin and Hilf,
1976). Briefly, samples treated with 10% metaphosphoric acid and centrifuged to eliminate the protein content. The GSH in the aqueous fraction so obtained was selectively reacted with 1%
OPT (dissolved in pure methanol) in sodium phosphate buffer (500 mM; pH 8.0).. The mixture was incubated for 15 min at room temperature and fluorescence was measured at 350/425nm.
For measuring protein sulfhydryl content, approximately 250µg of clear liver homogenate was reacted with 10mM DTNB (in methanol) in a 0.1M Tris buffer (pH 8.2) containing 10mM EDTA. The absorbance was recorded at 412nm after 10 min incubation. The

readings were adjusted for sample blank measurement where DTNB (5,5'-dithio-bis-(2nitrobenzoic acid)) was excluded in the reaction.
Sub-cellular fractionation of liver tissue
Isolation of nuclear, mitochondrial and cytosolic fraction from liver tissue was carried out according to the previously described method (Dimauro et al., 2012). Briefly, liver tissue, washed with cold PBS, was minced and thoroughly ground using pestle-mortar on ice and thereafter suspended in 5ml of STM buffer comprising 250 mM sucrose, 50 mM Tris–HCl (pH
7.4), 5 mM MgCl
2
, protease and phosphatase inhibitor cocktails. The homogenate was maintained on ice for 30 minutes, vortexed twice at maximum speed for 15 sec and then centrifuged at 800 g for 15 minutes. The supernatant was used for subsequent isolation of mitochondrial and cytosolic fractions while the pellet was resuspended in STM buffer and the
6 process was repeated until higher quality of nuclear isolates was detected under bright-field microscope. Further isolation of cytosolic and mitochondrial fractions, was done in exact accordance to the method detailed by Dimauro et al., 2012.
Nucleus Hoechst staining
The nuclei so obtained (as above) were incubated with 2µM Hoechst 33258 in 1X TBS for 15 min, rinsed and observed under Leica DMLB Fluorescence Microscope (Wetzlar, Germany) at
100X objective magnification.
Estimation of enzyme activities

i. SOD activity: SOD activity was assayed in the liver tissue lysate by the method of
Kakkar et al, 1984. The assay involved the addition of 90μl of 50mM sodium pyrophosphate
7 buffer (pH 8.3), to 10μl of liver tissue lysate, 30μl of 0.3mM nitro blue tetrazolium (NBT), followed by 10μl of 0.96 mM phenyl methyl sulphate and 40μl distilled water. The reaction was initiated by the addition of 20μl of 0.72mM NADH and the reaction was stopped after 90sec by the addition of 50μl glacial acetic acid. Absorbance was taken on SpectraMax Plus 384 at
560nm. Single unit of enzyme is expressed as 50% inhibition of NBT reduction/min/mg protein. ii. Catalase activity: Activity of catalase was assayed in fresh liver tissue lysates by taking 10ul of lysate and to it was added 100μl of 0.1M potassium phosphate buffer (pH 7.0) followed by 40μl of 2M H
2
O
2
, whereas respective blanks lacked H
2
O
2
. Instantly after 10sec, stopping reagent [5% Potassium dichromate and glacial acetic acid; (1:3)] was added and the absorbance was read at 530nm (Sinha et al; 1972).
Estimation of enzyme activities of glutathione reductase, thioredoxin reductase, glutathione peroxidase, NQO1 and glutathione-sulfotransferase was carried as per the procedure defined earlier (Rizvi et al., 2014).
Measurement of caspases activity
The activity of caspases namely, caspase-3, caspase-9 and caspase-12 were determined using the
Caspase-3 Colorimetric Assay Kit and Caspase-12 Fluorometric Assay Kit (BioVision, Inc.,
Mountain View, CA), respectively, as per the manufacturer’s instruction.
Immunoprecipitation and immunoblotting to detect Nrf2 ubiquitination
Analysis of ubiquitinated Nrf2 levels was done as described previously (Rizvi et al., 2014).

Immunoblot analysis
For in-vivo study, liver homogenates were prepared by grinding tissue in NP-40 lysis buffer using pestle-mortar. The suspension was centrifuged at 17,000g for 15 min. The
8 supernatant so obtained was collected and stored in aliquots at -80°C. Protein concentration was estimated using Bicinchoninic acid (BCA) method. 40-50 µg protein samples were resolved by
10% SDS-PAGE and thereafter transferred onto polyvinylidene fluoride (PVDF) membrane
(Hybond-P, Amersham Biosciences, Piscataway, NJ). Immunoblot was visualized using
Immobilon western chemiluminescent horseradish peroxidase substrate kit (Millipore
Corporation, Billerica, MA, USA). Chemiluminescence signals were captured using VersaDoc image analyzer (Bio-Rad, Hercules, CA, USA) and protein expression levels were analysed using ImajeJ 1.44p software (National Institute of Health, Bethesda, MD, USA).
Statistical Analysis
All computational calculations of quantitative data were performed using Microsoft Excel program. Each experiment was repeated at least three times. The quantitative variables represented in histograms are expressed as mean±SEM. Statistical comparisons between means of different groups were conducted by one-way Analysis of Variance (ANOVA) followed by
Tukey's post hoc test using SPSS 14.0 statistical package (SPSS Inc., Chicago, IL, USA).
Differences were considered statistically significant when P<0.05.
Supporting References

Dalle-Donne I, Rossi R, Giustarini D, Milzani A, Colombo R. Protein carbonyl groups as biomarkers of oxidative stress. Clin Chim Acta. 2003;329:23-38.
Dimauro I, Pearson T, Caporossi D, Jackson MJ. A simple protocol for the subcellular fractionation of skeletal muscle cells and tissue. BMC Res Notes. 2012;5:513.
Hedley DW, Chow S. Evaluation of methods for measuring cellular glutathione content using flow cytometry. Cytometry 1994;15:349-358.
Hissin PJ, Hilf RA. A fluorometric method for determination of oxidized and reduced glutathione in tissue. Anal.Biochem. 1976;74:214-225.
Janssen YM, Sen CK. Nuclear factor kappa B activity in response to oxidants and antioxidants.
Methods Enzymol. 1999;300:363-374.
Kakkar P, Das B, Viswanathan PN. A modified spectrophotometric assay of Superoxide dismutase. Indian J Biochem Biophys. 1984;2:130-132.
9
Niknahad H, Shuhendler A, Galati G, Siraki AG, Easson E, Poon R, O'Brien PJ. Modulating carbonyl cytotoxicity in intact rat hepatocytes by inhibiting carbonyl metabolizing enzymes. II.
Aromatic aldehydes. Chem Biol Interact. 2003;143-144:119-128.
Rizvi F, Shukla S, Kakkar P. Essential role of PH domain and leucine-rich repeat protein phosphatase 2 in Nrf2 suppression via modulation of Akt/GSK3β/Fyn kinase axis during oxidative hepatocellular toxicity. Cell Death Dis. 2014;5:e1153.
Sheehan D, Hrapchak B.Theory and practice of Histotechnology. 2nd Edition, 1980, 155-156,
Battelle Press, Ohio.

Sinha AK. Colorimetric assay of catalase. Anal Biochem. 1972;47: 389-394.
Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, et al.
Measurement of protein using bicinchoninic acid. Anal Biochem 1985;150:76-85.
10