Supplementary Materials (SM) for
Bisphenol A modulates colorectal cancer protein profile and promotes the metastasis via induction of epithelial to mesenchymal transitions
Zhuo-Jia Chen1*, Xiang-Ling Yang3, Hao Liu4, Wei Wei2,Kun-Shui Zhang5, Hong-Bin Huang1, John P. Giesy6, Huan-Liang Liu3,Jun Du2*, Hong-Sheng Wang2*
1 Department of Pharmacy, Sun Yat-sen University Cancer Center; State Key Laboratory of Oncology in South China; Collaborative Innovation Center for Cancer Medicine, Guangzhou 510060, China;
2 Department of Microbial and Biochemical Pharmacy, School of Pharmaceutical Sciences, Sun Yat-sen University, No. 132 Waihuandong Road, University Town, Guangzhou 510006, China;
3Guangdong Institute of Gastroenterology and the Sixth Affiliated Hospital; Institute of Human Virology; Key Laboratory of Tropical Disease Control (Ministry of Education), Sun Yat-sen University, Guangzhou, 510655,China
4Cancer Research Institute and Cancer Hospital, Guangzhou Medical University, Guangzhou 510095, China
5Department of Pharmacy, Sun Yat-sen Memorial Hospital of Sun Yat-sen University, Guangzhou 510120, China;
6 Department of Veterinary Biomedical Sciences & Toxicological Center, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
* Co-corresponding Authors: Dr ZJ Chen, Email: ; Dr HS Wang, Email: ; and Prof J DU, Email:.
Supplementary Materials Available
The detail procedures of two-dimensional electrophoresis (2-DE), protein visualization and image analysis, In-gel tryptic digestion of proteins and MALDI-TOF-MS/MS analysis, Western blotting analysis, transwell migration/invasion assay are stated. Two figures and one table were stated.
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MATERIALS AND METHODS
Two-dimensional electrophoresis (2-DE), protein visualization and image analysis
Control and treated cells were lysed in a lysis buffer (7 M urea, 2 M thiourea, 4% (v/v) CAHPS, 65 mM DTT, l mM PMSF, 0.5% (v/v) Bio-lyte, and 0.5% (v/v) IPG buffer) for 30 min at 4 °C. Samples were then centrifuged at 12,000 rpm for 30 min at 4 °C. After centrifugation, supernatants were collected, and protein concentrations were determined by Bio-Rad Dc protein assay. Isoelectric focusing (IEF) was carried out using the Bio-Rad PROTEAN IEF cell (Bio-Rad) and 17 cm Immobiline dry strips with a linear pH gradient of 3-10. Protein samples (1 mg) were loaded during the rehydration step (12 h). IEF was then performed at 20 °C in a stepwise manner: 200 V (1 h), 500 V (1 h), 1 kV (1 h), gradient 8 kV (0.5 h), and finally 8 kV for a total of 48,000 Vhs. Following IEF, the IPG strips were equilibrated for 15 min in equilibration buffer (50 mM Tris-HCl pH 8.8, 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, and 1% (w/v) DTT), then the gel strip was equilibrated once more in the same equilibration buffer containing 2.5% (w/v) iodoacetamide instead of DTT. The second dimensional SDS-PAGE was performed on 10% acrylamide gels, and electrophoresis was undertaken at 18 °C in Laemmli running buffer (20 mM Tris-HCl, 192 mM glycine, 0.1% SDS) using a PROTEIN II xi Multi-Cell system (Bio-Rad). Separation was carried out at 10 mA/gel for 1 h and 30 mA/gel overnight. After electrophoresis, gels were stained with Coomassie Brilliant Blue (CBB) G-250. To ensure data reliability, sample preparation (both control and treated cells) and 2-DE were performed in triplicate.
2-DE gels were scanned at 600 dpi resolution with a UMAX Power Look 2100XL scanner (Maxium Technologies, Taipei, China). Image analysis was performed with PDQuest version 7.3 (Bio-Rad, Hercules, CA, USA). The optimized parameters were as follows: saliency 2.0, partial threshold 4, and minimum area 50. The gel images were normalized according to the total quantity in the analysis set. This normalization method provided by PDQuest software divided each spot abundance value by the sum of total spot abundance values to obtain individual relative spot abundances. To study changes in expression in more detail, patterns for control samples were set as references to be compared qualitatively and quantitatively with those of cells treated with 10-8 M or 10-5 M BPA. Relative comparison of the intensity abundance between control and treated cells (3 replicate samples for each group) were performed using the Student’s test after checked for normality and homogeneity of variance. Expression intensity larger than 2.0 (p ≤ 0.05) or smaller than 0.5 (p ≤ 0.05) were set as thresholds indicating significant changes.
In-gel tryptic digestion of proteins andMALDI-TOF-MS/MS analysis
Protein spots were manually excised from CBB-stained gels and transferred to V-bottom 96-well microplates containing 100 μL of 50% acetonitrile/25 mM ammonium bicarbonate solution per well. After being destained for 1 h at 40 °C, gel plugs were dehydrated with 100 μL of 100% acetonitrile for 20 min and then thoroughly dried in a SpeedVac concentrator (Thermo Fisher Scientific, Waltham, MA) for 30 min. The dried gel particles were rehydrated at 4 °C for 45 min with 10 ng sequencing grade modified trypsin (Promega, Madison, WI, USA) dissolved in 25 mM ammonium bicarbonate and then incubated at 37 °C for 12 h. After trypsin digestion, the peptide mixtures were extracted with 8 μL of extraction solution (50% acetonitrile/0.5% trifluoroacetic acid) per well at 37 °C for 1 h. Finally, extracts were dried under nitrogen gas.
Peptides were eluted with 0.8 μL matrix solution (α-cyano-4-hydroxy-cinnamic acid in 0.1% trifluoroacetic acid, 50% acetonitrile) before application to the target plate. Samples were allowed to air-dry and analyzed using an Ultraflex II MALDI-TOF/TOF mass spectrometer (Bruker Daltonics, Bremen, Germany). The UV laser was operated at a 200 Hz repetition rate with a wavelength of 355 nm. The accelerated voltage was operated at 25 kV. MS spectra were acquired over a mass range of 800-5000m/z. Peptide Calibration Standard II (Bruker Daltonics) was used to calibrate the mass instrument with internal calibration mode. From the PMF for each spot the 10 largest peaks, with a signal-to-noise threshold > 30, were automatically selected for MS/MS fragmentation in LIFT mode without addition of a collision gas. Flex Analysis software (version 3.3, Bruker Daltonics) was used to perform the spectral processing and peak list generation for both MS and MS/MS spectra. Processed peak lists were submitted to Mascot (version 2.3, Matrix Science, , London, U.K.) via the Biotools 3.1 (Bruker Daltonics) and searched using the following parameters: NCBInr database, taxonomy of Homo sapiens (human), trypsin of the digestion enzyme, one missed cleavage site, carbamidomethylation (C) as a fixed modification and oxidation (M) as a variable modification, MS tolerance of 100ppm, MS/MS tolerance of 0.6 Da, and the significance threshold was set at p0.05.
Transwell migration/invasion assay
The polycarbonate filters (8 μm pore size, Corning) pre-coated with Matrigel Matrix (BD Biosciences) were used for invasion assay, and uncoated filters were used for migration assay. Cells (1×105) in 300 μl medium (containing 0.1% FBS) with or without BPA were seeded in the upper chamber. Then 600 μl medium with 10% FBS was added to the lower chamber and served as a chemotactic agent. After 48 h incubation, for migration, the cells migrated and adhered onto the lower chamber were fixed in 4% paraformaldehyde for 20 min, stained with hematoxylin and counted under upright microscope (5 fields per chamber). To quantify invasion, cells in the upper chamber were fixed in 4% paraformaldehyde for 20 min. Then the Matrigel was mechanically removed from the filter with a cotton swab. Cells adhering to the under-side of the filter were stained with hematoxylin and counted under upright microscope (5 fields per chamber). Each migration and invasion assay was repeated in three independent experiments.
Western blotting analysis
Briefly, cells were lysed in cell lysis buffer, and then lysates were cleared by centrifugation and denatured by boiling in Laemmli buffer. Aliquats of protein were separated on 10% sodium dodecyl sulfate (SDS)–polyacrylamide gels and electrophoretically transferred to nitrocellulose membranes. Following blocking with 5% non-fat milk at room temperature for 2 h, membranes were incubated with the primary antibody at 1:1000 dilution overnight at 4 °C and then incubated with a horseradish peroxidase-conjugated secondary antibody at 1:5000 dilution for 2 h at room temperature, and detected with the Western Lightning Chemiluminescent detection reagent (Perkin-Elmer Life Sciences, Wellesley, MA).
Figure legends
Fig. S12-DE analysis of expression of proteins in SW480 cells exposed BPA.Cells were treated with 10-8 M or 10-5 M BPAfor 48 h. Total proteins (1 mg) were loaded on a 17 cm IPG strip with linear gradient (pH 3-10) and SDS-PAGE was performed on a 10% gel. Proteins were stained with CBB G-250. Proteins indicated by arrow were differentially regulated according to treatment and identified by MS and MS/MS, numbers are correlated with these spots no. listed in Table 1.
Fig. S2 Validation of differentially expressed proteins. Close-up of selective differential expression protein spots in Figure S1 (A) and Western blot analysis of the selective differential expression proteins in SW480 cell (B), LoVo cell (C), and HCT 116 cell (D).
Fig.S1
Fig.S2
1
Table S1．Identification of differentially expressed proteins in SW480 cells treated with BPA by MALDI-TOF- MS/MS
Spot no. a / Protein name / Gene symbol / Accessionno. b / MW
(kDa)c / pI d / Matched
Peptides / Protein
Score e / Coverage
(%) f / % change (Treated*100/Control)g
Low / High
Cell structure and motility
2 / lamin-B1 isoform 1 / LMNB1 / gi|5031877 / 66653 / 5.11 / 45 / 875 / 63 / 131±4.37 / 170±3.36
6 / T-complex protein 1 subunit alpha isoform a / TCP-1 / gi|57863257 / 60819 / 5.80 / 31 / 543 / 58 / 135±2.52 / 97.9±3.48
13 / fascin / Fascin / gi|4507115 / 55123 / 6.84 / 27 / 882 / 63 / 161±1.24 / 185±1.08
16 / T-complex protein 1 subunit zeta isoform a / TCP1Z / gi|4502643 / 58444 / 6.23 / 27 / 610 / 59 / 264±5.37 / 245±5.80
22 / T-complex protein 1 subunit delta isoform a / TCP1D / gi|38455427 / 58401 / 7.96 / 23 / 506 / 42 / 54.0±1.98 / 183±5.84
30 / protein tyrosine kinase 9-like / PTK9L / gi|48145581 / 39779 / 6.37 / 15 / 307 / 51 / 94.7±0.54 / 94.4±0.54
45 / annexin A1 / Annexin A1 / gi|119582950 / 40475 / 6.57 / 18 / 522 / 63 / 226±2.43 / 301±10.8
25 / annexin A2 isoform 2 / annexin A2 / gi|4757756 / 38808 / 7.57 / 25 / 752 / 55 / 207±8.54 / 316±5.58
53 / tropomyosin alpha-3 chain isoform 2 / TMα / gi|24119203 / 29243 / 4.75 / 14 / 158 / 45 / 186±1.08 / 162±1.24
64 / Keratin 8 / Krt8 / gi|49256423 / 53774 / 5.52 / 26 / 750 / 61 / 50.9±4.80 / 84.4±2.95
23 / aspartate aminotransferase / AST/AAT / gi|4504067 / 46447 / 6.52 / 21 / 801 / 65 / 207±8.54 / 219±4.02
36 / proteasome subunit alpha type-6 / Psma6 / gi|8394076 / 27838 / 6.34 / 17 / 471 / 47 / 55.4±9.05 / 41.1±12.2
Cell proliferation, cell cycle, and apoptosis
29 / translation initiation factor / TIF4A / gi|496902 / 47088 / 6.08 / 19 / 304 / 43 / 577±8.79 / 491±10.3
31 / eukaryotic translation initiation factor 3 subunit H (EIF3H protein) / EIF3H / gi|60688399 / 39756 / 5.96 / 13 / 172 / 20 / 207±4.23 / 154±5.71
34 / omega-amidase NIT2 / NIT2 / gi|9910460 / 30988 / 6.82 / 13 / 602 / 45 / 103±9.25 / 0.40±0.04
40 / heat shock protein 27 / Hsp27 / gi|662841 / 22427 / 7.83 / 7 / 286 / 41 / 182±4.43 / 93.2±8.65
48 / GTP-binding nuclear protein Ran / GTPase Ran / gi|5453555 / 24579 / 7.01 / 14 / 387 / 50 / 110±6.60 / 182±4.01
52 / SET / SET / gi|145843637 / 26593 / 4.73 / 9 / 432 / 28 / 201±0.23 / 226±0.20
68 / voltage-dependent anion-selective channel protein 2 isoform 1 / VDAC-2 / gi|296317337 / 33864 / 7.51 / 13 / 699 / 58 / 151±3.09 / 150±5.19
Energy metabolism
4 / myo-inositol 1-phosphate synthase A1 / mIPS A1 /
gi|119605112 / 45271 / 5.55 / 16 / 213 / 42 / 90.7±8.12 / 174±4.23
11 / glucose-6-phosphate dehydrogenase / G6PD / gi|26224790 / 55188 / 6.91 / 37 / 775 / 65 / 385±7.06 / 154±4.39
14 / aldehyde dehydrogenase family 1 member A3 / ALDH 1 / gi|153266822 / 56871 / 6.99 / 16 / 424 / 36 / 206±4.42 / 183±4.97
15 / glutamate dehydrogenase 1 / GLUD1 / gi|20151189 / 56315 / 6.71 / 34 / 793 / 55 / 207±4.29 / 260±3.41
18 / Chain A, The Structure Of Human Transketolase / TKTL1 / gi|301598628 / 67733 / 7.57 / 24 / 637 / 37 / 60.0±6.30 / 178±5.50
26 / phosphoserine aminotransferase isoform 1 / PSAT1 / gi|17402893 / 40796 / 7.56 / 20 / 317 / 49 / 165±1.23 / 143±1.41
28 / Chain A, Crystal Structure Of Human Enolase 1 / Enolase 1 / gi|203282367 / 47350 / 6.99 / 27 / 480 / 55 / 71.7±5.66 / 50.5±8.04
32 / malate dehydrogenase, cytoplasmic isoform 3 / MDH / gi|312283703 / 27179 / 9.18 / 7 / 252 / 31 / 129±2.43 / 136±2.30
35 / Triosephosphate isomerase 1 / TPI / gi|17389815 / 26910 / 6.45 / 13 / 324 / 50 / 95.1±2.76 / 70.1±3.75
38 / alpha-enolase isoform 1 / Enolase-1 / gi|4503571 / 47481 / 7.01 / 22 / 795 / 53 / 115±2.97 / 73.8±4.65
59 / fumarate hydratase, mitochondrial / FH / gi|19743875 / 54773 / 8.85 / 21 / 899 / 53 / 134±2.90 / 146±2.67
66 / Human Heart L-Lactate Dehydrogenase H / LDH / gi|13786847 / 36769 / 5.72 / 28 / 540 / 58 / 71.8±6.48 / 89.5±5.20
67 / triosephosphate isomerase isoform 1 / TPI / gi|4507645 / 26938 / 6.45 / 12 / 653 / 58 / 81.7±3.09 / 70.3±5.19
Oxidative stress
3 / NADH-ubiquinone oxidoreductase 75 kDa subunit, mitochondrial isoform 4 / complex I / gi|316983156 / 74393 / 5.61 / 33 / 726 / 49 / 97.0±7.27 / 173±4.07
7 / cytochrome b-c1 complex subunit 1, mitochondrial precursor / UQCRC1 / gi|46593007 / 53297 / 5.94 / 29 / 687 / 62 / 113±1.18 / 115±4.17
9 / stomatin-like protein 2 / STOML2 / gi|7305503 / 38624 / 6.88 / 11 / 738 / 42 / 117±5.15 / 170±3.53
20 / ATP synthase subunit alpha, mitochondrial isoform c / ATPase C / gi|50345982 / 54574 / 8.24 / 26 / 748 / 48 / 76.9±9.80 / 249±6.12
21 / ATP synthase subunit alpha, / ATPase A / gi|4757810 / 59828 / 9.16 / 23 / 601 / 44 / 99.7±7.76 / 184±4.21
24 / multifunctional protein ADE2 isoform 2 / ADE2 / gi|5453539 / 47790 / 6.95 / 23 / 816 / 47 / 141±12.6 / 216±8.26
33 / methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 2, methenyltetrahydrofolate cyclohydrolase / MTHFD1 / gi|119620079 / 36115 / 8.70 / 12 / 571 / 46 / 127±2.30 / 176±1.66
37 / Coproporphyrinogen oxidase / CPO / gi|23272125 / 50932 / 8.59 / 27 / 691 / 58 / 138±2.48 / 103±3.33
43 / peroxiredoxin 3 / PRDX-3 / gi|119569783 / 11158 / 6.06 / 9 / 543 / 94 / 238±4.93 / 135±8.73
46 / protein DJ-1 / DJ-1 / gi|31543380 / 20050 / 6.33 / 13 / 559 / 68 / 647±9.46 / 388±2.43
50 / Human Mitochondrial Acetoacetyl-Coa Thiolase / ACAT1 / gi|83755034 / 42867 / 7.71 / 8 / 312 / 20 / 89.1±5.78 / 148±3.47
51 / cytochrome b-c1 complex subunit 2 / UQCRC2 / gi|50592988 / 48584 / 8.74 / 18 / 856 / 38 / 85.2±10.2 / 184±4.73
Protein folding, metabolism, and modification
42 / endoplasmic reticulum resident protein 29 / ERp29 / gi|5803013 / 29032 / 6.77 / 11 / 210 / 32 / 171±3.60 / 113±5.44
54 / heat shock cognate 71 kDa protein / Hsp 70 / gi|5729877 / 71082 / 5.37 / 28 / 676 / 43 / 79.2±8.19 / 23.2±2.94
62 / ER-60 protease / ER-60 protease / gi|1208427 / 57160 / 5.98 / 31 / 981 / 59 / 85.6±6.71 / 121±4.72
63 / MTHSP75 / MTHSP75 / gi|292059 / 74019 / 5.97 / 22 / 700 / 35 / 102±13.0 / 151±8.82
1 / heterogeneous nuclear ribonucleoprotein K / hnRNP K / gi|119583084 / 49002 / 5.46 / 22 / 568 / 47 / 36.4±4.27 / 30.2±7.18
12 / Dihydrolipoamide Dehydrogenase / DLD / gi|83753870 / 50656 / 6.50 / 20 / 554 / 46 / 123±2.27 / 134±2.10
44 / elongation factor 1-beta / EF1B / gi|4503477 / 24919 / 4.50 / 10 / 529 / 46 / 78.1±0.82 / 101±0.64
DNA/RNA processing
17 / HNRNPL protein / HNRNPL / gi|211828181 / 62515 / 7.07 / 25 / 296 / 46 / 128±2.27 / 175±1.65
19 / Chain A, structure of human muscle pyruvate kinase (Pkm2) / Pkm2 / gi|67464392 / 60277 / 8.22 / 37 / 928 / 64 / 114±7.12 / 214±3.82
39 / prohibitin / PHB / gi|4505773 / 29843 / 5.57 / 15 / 788 / 79 / 128±3.29 / 152±2.79
Signal transduction
5 / peptidyl-prolyl cis-trans isomerase FKBP4 / FKBP4 / gi|4503729 / 52057 / 5.35 / 25 / 654 / 56 / 137±2.22 / 172±1.76
41 / 14-3-3 protein epsilon / YWHAZ / gi|5803225 / 29326 / 4.63 / 17 / 477 / 55 / 319±3.11 / 360±2.76
aSpot numbers correspond with 2-DE gel as shown in Figure S1. b Accession number in NCBInr database. c MW (kDa): molecular mass of predicted protein. d pI: pI of predicted protein. e Protein score: In MASCOT, the score for an MS/MS match is based on the absolute probability (P), and the observed match between the experimental data and database sequence is a random event. The reported score is -10 log (P). So during a search, if 1.5×105 peptides fell with the mass tolerance window the precursor mass, and the significance threshold was chosen to be 0.05, this would translate into a score threshold of 65. f Percentage of predicated protein sequence covered by matched sequence. g Fold change is expressed as a radio of the Vol % between of treated/control cells, and each value represents the mean value ±SD of three independent experiments.
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