Cell culture
Human colorectal cancer cell line, HT29, was obtained from Pasteur Institute (Tehran, Iran). This cell line was cultured in RPMI-1640 medium (Gibco) and supplemented with 10% FBS (Gibco) and 100 U/ml of penicillin-streptomycin (Sigma). Cells were grown in standard condition (5% CO2, 37 °C and humidified atmosphere). The medium was changed every 3 days and passaged using 0.25% Trypsin–0.01% EDTA. For all experiments, under sixth passage cells were used.
Generation of NFE2L2-knocked down cell line
In order to knock down Nrf2, we used Santa Cruz Nrf2 shRNA plasmid (sc-37,049-SH) and used the manufacturers’ protocol. Briefly, 1.2 × 105 cells were seeded in 12 well plate and waited until cells reached to 70% confluence. Subsequently, 1 μg of plasmid and 4 μl TurboFect Transfection Reagent (Thermo Scientific; R0533) were diluted in 100 μl serum-free DMEM. After 15 minutes, the DNA/reagent complex was added to the wells drop wise and incubated in standard condition for next 48 h. Then, the cells were exposed to puromycin selection media for 14 days. To confirm the gene knock down, real time PCR was used (HT29- Nrf2−). In addition, shRNA plasmid alone transfection was used as a mock control (Santa Cruz; sc-108,060) [10].
RNA extraction and real time PCR
Total RNA was extracted from the cells by spin column kit (Favorgen; FABRK 001) and its quality was examined (A260/280 ratio ≥ 1.9). One microgram of total RNA was used for cDNA synthesis (by PrimeScript; 6110B). qPCR reaction was carried out by using SYBR Green PCR Master Mix in Step one Real Time PCR instrument (Applied Biosystems) in 95 °C/5 min for initial denaturation then 95 °C/10s, 58 °C/30s and 72 °C/30s for 40 cycles. Analysis carried out by 2-ΔΔCt method with Beta-2 Microglobulin (β2M) as internal control. Primers were designed by PerlPrimer software as follows;
5´-F, TTCCCGGTCACATCGAGAG-3´, 5´-R, TCCTGTTGCATACCGTCTAAATC-3´ for NFE2L2, 5´-F, GACCCATGACACCAAGGA-3´, 5´-R, GCTGAGTGTAAGGACCCA-3´ for HO-1 and 5´-F, AGGCTATCCAGCGTACTCC-3´, 5´-R, ATGTCGGATGGATGAAACCC-3´ for β2M.
Viability and metastatic properties
Cellular cytotoxic response against two different chemotherapy drug, doxorubicin and cisplatin, was evaluated by MTT (methylthiazol tetrazolium bromide) assay. 2 × 104 Cells were plated in 96 well cell culture plate containing 200 μl 10% FBS media. After 24 or 48 h, different concentration of drugs were subjected to HT29 and HT29-Nrf2−cell lines. Twenty microliter of MTT (5 mg/ml) (Sigma) was added to each and incubated for 4 h in standard condition. Finally, the cells supernatant were changed with DMSO and absorbance read at 570 nm (with reference wavelength of 630 nm) in ELISA plate reader (BioTek).
The metastasis properties of cells were investigated using the scratch assay. After reaching desired confluency, a monolayer of cells was scratched to create a cell-free line by a yellow tip, and then cells were treated with 0.6 μM of doxorubicin and 125 μM cisplatin for 24 and 48 hours. Distances of two edges were measured using ImageJ 1.48 software [11].
Non enzymatic and enzymatic antioxidant assay
To measure antioxidant and prooxidant balance, cells were lysed by 0.5 ml of ice-cold lysis buffer (Cat No: FNN001; Invitrogen) and the bradford method were done to measure the total protein content. Then commercially available colorimetric kit was used to determine the malondialdehyde (MDA) (ZellBio; MDA48), total antioxidant capacity (TAC) (Randox; NX2332) based on ABTS (Azino ethylbenzthiazoline sulphonate) oxidation, super oxide dismutase (SOD) (ZellBio; SOD48), glutathione peroxidase (GPX) (ZellBio; GPX48), catalase (CAT) (ZellBio; CAT48), total glutathione (GSH) (Sigma; CS0260) and H2O2 (Sigma, MAK165) assay according to the manufacturer’s instruction.
Nano-flow LC-MS/MS proteomic procedure
In bottom-up proteomics approaches, Nanoflow liquid chromatography combined with high-resolution mass spectrometry (Nano-flow LC-MS/MS), is 100 times more sensitive than traditional LC-MS. Low column internal diameter (ID; 75 μM) makes it a useful technique for identification of cellular proteome changes [12].
Protein extraction and digestion
After lysing cells in 8 M urea/0.1 M Tris-HCl, pH 8.0 with protease inhibitor cocktail (Roche), extracted proteins were reduced in 10 mM DTT (Dithiothreitol) for 2 h and alkylated in 20 mM iodoacetamide for 30 min at 25 °C in dark condition. Then, the protein solution was diluted 1:5 with 50 mM TEAB (triethylammonium bicarbonate) and digested overnight with trypsin (1:50 ratio) at 37 °C. At the end, the digestion was quenched via acidification with formic acid. After that, we used OASIS HLB column to desalt the digestion, and finally peptides were eluted with 60% acetonitrile and lyophilized via vacuum centrifugation.
Peptide labeling with TMT
Prior to Tandem Mass Tag (TMT) labeling, the dried peptide was resolved in TEAB (Triethylammonium bicarbonate buffer) and then 100 μg protein of each biological replicates were labeled with TMT-130 and TMT-131 (Thermo Scientific) according to the manufacturer’s protocol.
Peptides fractionation by pH reverse phase chromatography
For desalting the mixed TMT-labeled peptides, Sep-Pak Vac C18 SPE cartridges (Waters, Massachusetts, USA) was used following drying in a vacuum concentrator. Next, this peptide suspended in 2% acetonitrile, pH 10 (solution A) and loaded onto YMC-Triart C18 basic RP-LC column (250 × 4.6 mm, 5 um particles). For peptide separation, a binary buffer of solution A and B (98% acetonitrile) operated at 0.7 ml/min on L-3000 HPLC System (Rigol). All fractions were collected at 90s intervals and concatenated into 12 post-fractions and lyophilized until nanoLC-MS/MS analysis.
LC-MS/MS analysis
All nanoLC-MS/MS experiments were performed on a Q Exactive (Thermo Scientific) equipped with an Easy n-LC 1000 HPLC system (Thermo Scientific). The labeled peptides were loaded onto a 100 μm id× 2 cm fused silica trap column packed in-house with reversed phase silica (Reprosil-Pur C18 AQ, 5 μm, Dr. Maisch GmbH) and then, separated on a 75 μm id× 20 cm C18 column packed with reversed phase silica (Reprosil-Pur C18 AQ, 3 μm, Dr. Maisch GmbH). The peptides bounded on the column were eluted with a 78 min linear gradient. The solvent A consisted of 0.1% formic acid in water solution and the solvent B consisted of 0.1% formic acid in acetonitrile solution. The segmented gradient was 5–8% B, 8 min; 8–22% B, 50 min; 22–32% B, 12 min; 32-95% B, 1 min; 95% B, 7 min at a flow rate of 310 nl/min.
The MS analysis was performed with Q Exactive mass spectrometer (Thermo Scientific). With the data-dependent acquisition mode, the MS data were acquired at a high resolution 70,000 (m/z 200) across the mass range of 300–1600 m/z. The target value was 3.00E+ 06 with a maximum injection time of 60 ms. The top 20 precursor ions were selected from each MS full scan with isolation width of 2 m/z for fragmentation in the HCD collision cell with normalized collision energy of 32%. Subsequently, MS/MS spectra were acquired at resolution 17,500 at m/z 200. The target value was 5.00E+ 04 with a maximum injection time of 80 ms. The dynamic exclusion time was 40s. For nano-electrospray ion source setting, the spray voltage was 2.0 kV; no sheath gas flow; the heated capillary temperature was 320 °C.
The Proteome Discovery (V; 2.2.0.388) was used for protein identification and Percolator for false discovery rate (FDR) analysis of raw data from Q Exactive. For searching of human protein database, we used Uniprot search engine and set some parameters. Trypsin was selected as the enzyme and two missed cleavages were allowed; the mass tolerance of precursor was set to 10 ppm and the product ions tolerance was 0.02 Da.; TMT 2plex (lysine and N-terminus of peptides) and cysteine carbamidomethylation were chosen as fixed modifications and the methionine oxidation was specified as variable modification. The peptides confidence was set as high for peptides filter and FDR < 1% was set for protein identification. Proteins quantification was performed using the ratio of the intensity of reporter ions from the MS/MS spectra. Unique and razor peptides were taken for protein relative quantification. The co-isolation threshold was specified as 50% and average reporter’s S/N value should be above 10. The normalization to the protein median of each sample was used to correct experimental bias and the normalization mode was selected as total peptide amount.
Heatmap visualization and functional annotation
Hierarchical clustering with Pearson’s correlation of the differently expressed proteins were analyzed by R software. For gene ontology determination, differently expressed proteins were divided into up and down regulated proteins that were then categorized into three groups MF, CC and BP (Molecular function, Cellular component and Biological process). Along with this procedure KEGG enriched pathways were determined by using human Enrich r database [13, 14].and the important terms ranked based on Fisher’s exact test calculated p-value. All sections in gene annotation were grouped based on their gene ontology number and p-value < 0.05 [15].
Protein-protein interaction (PPI) network
String webserver (https://string-db.org/) is the most well-known online database for simple and more rapid technique to assess PPI. 172 differently expressed proteins were uploaded and the minimum required interaction score were set to high confidence (0.7). Experiments, databases and text mining were used as active interaction sources. The TSV file was visualized in Cytoscape software (version 3.7.1). In graph theory, the number of connections to each node was defined as degree. The betweenness value for each node was related to its centrality in clusters.
Western blot
Total proteins were extracted from HT29 and HT29-Nrf2− cells with RIPA cell lysis buffer (Cell Signaling) on ice and quantified using Bradford standard technique. One hundred microgram of extracted proteins loaded onto 10% SDS-PAGE and transferred to PVDF membrane. Then PVDF was blocked with 5% BSA for 2 h and then incubated with primary antibodies ERK 1/2 (Santa Cruz, USA, #sc-292,838), Heme Oxygenase 1 (HO-1), (Santa Cruz, USA, #sc-136,960), Ki-67 (Santa Cruz, USA, #sc-23,900), PKLR (Santa Cruz, USA, #sc-166,228), SLC25A27 (UCP4) (Mybiosource, USA, #MBS668834), FOXO3a (Cell Signaling, USA, #2497), cytochrome c (Cyt c) (Santa Cruz, USA, #sc-13,156) and β-actin (Santa Cruz, USA, #sc-130,301) overnight at 4 °C. After several washing with TBST, the PVDF was incubated with secondary mouse IgG (Santa Cruz, USA, #516102) or mouse anti-rabbit (Santa Cruz, USA, #sc-2357) for 2 h at 37 °C. The protein bands were visualized using ECL Detection reagent (Pierce, Rockford, IL, USA). Densitometry band quantification was performed using ImageJ software.
Statistical analysis
All experiments were performed in triplicate and data were expressed as mean ± SD. Student’s t-test was used to assess the significance of differences in experiments with only two groups and one-way Analysis of variance (ANOVA) was used in experiments with more than two groups. We performed data analysis with GraphPad prism software version 2.02. Statistical differences between groups are indicated as follows: *P < 0.05, **P < 0.01, and ***P < 0.001.