Differential Expression of Peroxisomal Proteins in Distinct Types of Parotid Gland Tumors
Abstract
:1. Introduction
2. Results
2.1. Pathology of Parotid Tumor Entities Using HE Staining
2.2. Myoepithelial and Acinar Cell Markers in Parotid Tumor Entities
2.3. Differential Expression of Peroxisomal Biogenesis, Matrix Proteins and Enzymes in Parotid Tumors
2.3.1. Peroxisomal Biogenesis Proteins
2.3.2. Peroxisomal β-Oxidation Enzymes of Pathway 1 and 2
2.3.3. Plasmalogen and Cholesterol Synthesis Enzymes
2.3.4. Catalase, Superoxide Dismutase and Extra-Peroxisomal Antioxidative Enzymes
2.4. Peroxisome Proliferator-Activated Receptors (PPARs)
3. Discussion
4. Materials and Methods
4.1. Tissue Harvesting and Preparation
4.2. Paraffin Embedding and Antigen Retrieval
4.3. Hematoxylin and Eosin (HE) Staining
4.4. Immunofluorescence (IF) Staining
4.5. RNA Extraction and Isolation
4.6. Quantitative Reverse Transcriptase Polymerase Chain Reaction (qPCR)
4.7. Statistical Methods and Tools
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Primary Antibody | Host | Target Molecular Weight | Dilution (IF) | Dilution (WB) | Supplier |
---|---|---|---|---|---|
Cell type specific antigens | |||||
Parotid secretory Protein, mouse (SPLUNC2) | Goat, polyclonal | 25 kDa | - | 1:3000 | Acris Antibodies, Inc. Cat. no.: AP31965PU-N |
Parotid secretory Protein, human (SPLUNC2) | Mouse, monoclonal | 25 kDa | 1:1000 | Abbexa, Cat. no.: abx11413 | |
Peroxisomal biogenesis and metabolic proteins | |||||
Peroxin 6 (PEX6p), | Rabbit, polyclonal | 104 kDa | 1:1000 | Novus Biologicals, cat. no.: NBP1-80955 | |
Peroxin 7 (PEX7p), | Mouse, monoclonal | 40 kDa | 1:1000 | UC Davis/NIH NeuroMab Facility | |
Peroxin 13 (PEX13p), mouse | Rabbit, polyclonal | 44 kDa | 1:1000 | 1:6000 | Gift from Denis I. Crane; School of Biomol. Biophys. Sci., Griffith Univ., Nathan, Brisbane, Australia; see reference: [63] |
Peroxin 14 (PEX14p), mouse | Rabbit, polyclonal | 42 kDa | 1:1000 | 1:3000 | Gift from Denis I. Crane; see reference: [63] |
Catalase (CAT), mouse | Rabbit, polyclonal | 60 kDa | 1:2000 | 1:5000 | Gift from Denis I. Crane, see reference: [63] |
ABC-transporter D3 (ABCD3/PMP70), mouse | Sheep, polyclonal | 75 kDa | 1:1000 | 1:3000 | Gift from Stephen Gould, Johns Hopkins Univ., Dept. Biol. Chem., Baltimore, MD, USA; see reference: [64] |
Acyl-CoA oxidase (ACOX 1), mouse | Rabbit, polyclonal | 51 kDa | 1:5000 | Gift from Paul P. van Veldhoven, Dept. of Molecular Cell Biology, Pharmacology, Catholic University Leuven, Belgium; see reference: [65] | |
Peroxiredoxin 1 | Rabbit, polyclonal | 22 kDa | 1:1000 | Abcam, Cambridge, UK, Cat. no: ab59538 | |
“SKL”, mouse peptide | Rabbit, polyclonal | 1:5000 | Gift from Denis I. Crane, see reference: [63] | ||
Thiolase | Rabbit, polyclonal | 51 kDa | 1:1000 | 1:5000 | Gift from Nancy E Bravermann; Depts. of Human Genetics and Pediatrics, McGill University-Montreal Montreal, QC, Canada. |
Alkylglycerone-phosphate synthase (AGPS) | Mouse, monoclonal | 78 kDa | 1:1000 | 1:500 | Santa cruz, Cat no: sc-374201 |
Glyceronephosphate O-Acyltransferase(GNPAT) | Rabbit, polyclonal | 70 kDa | 1:500 | 1:5000 | Proteintech, Cat no: 14931-1-AP |
Nuclear receptors and cell signalling molecules | |||||
PPAR α, rabbit | Rabbit, polyclonal | 52 kDa | 1:1000 | Santa cruz, Cat no: sc-9000 | |
PPAR α | Mouse, monoclonal | 52 kDa | 1:1000 | Millipore, Cat. no.: MAB3890 | |
PPAR α | Mouse, monoclonal | 52 kDa | 1:1000 | Pierce Biotechnology, Cat. no.: MA 1--822 | |
PPAR β, rabbit | Rabbit, polyclonal | 52 kDa | 1:50 | 1:1000 | Santa cruz, Cat no: sc-7197 |
PPAR δ | Rabbit, polyclonal | 50 kDa | 1:1000 | Abiocode, Cat. no.: R2295-1 | |
PPAR γ, rabbit | Rabbit, polyclonal | 55 kDa | 1:50 | 1:1000 | Santa cruz, Cat no: sc-7196 |
Antioxidative enzymes from other cell compartments | |||||
Glutathione reductase | Rabbit, polyclonal | 56 kDa | 1:1000 | Abcam, Cambridge, UK, Cat. no: ab16801 | |
Oxidative phosphorylation complex III (OxPhosIII), human | Mouse, monoclonal | 1:1000 | Molecular Probes/Invitrogen, Carlsbad, USA, Cat. no.: A11143 | ||
Thioredoxin-1 (TrxR1) | Mouse, monoclonal | 55 kDa | 1:1000 | Santa cruz, Cat no: sc-28321 | |
Superoxide dismutase 1 (SOD-1), | Goat; polyclonal | 17 kDa | 1:5000 | R&D Systems, Cat. no.; AF3787 | |
Superoxide dismutase 2 (SOD-2) | Rabbit, polyclonal | 25 kDa | 1:1000 | 1:1000 | Research diagnostics, Inc., NJ, USA, Cat no: RDI-RTSODMabR |
HO-1 | Rabbit, polyclonal | 32 kDa | 1:1000 | 1:1000 | Stressgen, Cat no: SPA-895 |
Other marker proteins of different cell compartments | |||||
β-tubulin, | Mouse, monoclonal | 55 kDa | 1:10,000 | Sigma Aldrich, Inc., Cat. no.: T8328 | |
β-actin | Mouse, monoclonal | 45 kDa | 1:10,000 | Cell Signaling Technology, Inc.#3700 | |
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) | Mouse, monoclonal | 36 kDa | 1:60,000 | Hy Test Ltd., Cat. no.:5G4 | |
Secondary Antibodies | |||||
HRP-rabbit | 1:6000 | Cell Signaling Technology, Inc.# | |||
HRP-mouse | 1:6000 | Cell Signaling Technology, Inc.# | |||
Bovine anti goat HRP | 1:5000 | Santa cruz, Cat. no: sc-2378 |
Gene Target | Gene Bank Accession No. | Sence Primer (5′-3′) | Antisense Primer (5′-3′) | Annealing Temp °C | PCR Product (bp) |
---|---|---|---|---|---|
ABCD1 | NM_000033.3 | GTGGAGGACATGCAAAGGAA | TCACACATAGCCTCCCAACC | 58.1 | 113 |
ABCD3 | NM_002858.3 | ATGACCCTTGGAACACTTCG | TGCCATCCATATGCAGGTAG | 57.8 | 385 |
ACOX1 | NM_004035.6 | ATTTCCTTCAGGGGAGCATC | GCCAAGTGTCACATCCTGAA | 57.3 | 137 |
ACOX2 | NM_003500.3 | CAAATTGTCGGCCTCCTGTA | GAGATCTCTGTGGCGTGGAG | 57.9 | 125 |
ACOX3 | NM_003501.2 | GGAGTGTGTGGGCTCTTATC | CTCTTGCTCGGTAGGCATC | 57.7 | 107 |
ACTB | NM_001101.3 | TCCCTGGAGAAGAGCTACGA | AGCACTGTGTTGGCGTACAG | 59.4 | 194 |
AGPS | NM_003659.3 | AGGGGGATCGTGAGAAGGT | CCAAAGCCAAGTCTCGAATG | 59.6 | 147 |
CAT | NM_001752.3 | CGTGCTGAATGAGGAACAGA | TTGTCCAGAAGAGCCTGGAT | 57.9 | 150 |
FDPS | NM_001135821.1 | CAAGGAGGTCCTGGAGTACAA | GGAGACTATCAGCATCCTGTTTC | 58.7 | 113 |
GAPDH | NM_002046.5 | GTCAACGGATTTGGTCGTATT | TGTAGTTGAGGTCAATGAAGGG | 56.6 | 106 |
GNPAT | NM_014236.3 | GTGCAGAAAAACGCCTTAGC | GGCTGGTTTTCCTATTGGTG | 58.3 | 150 |
GPX1 | NM_000581.2 | CAGTTTGGGCATCAGGAGAA | TCGAAGAGCATGAAGTTGGG | 57.8 | 101 |
GR1/GSR | NM_000637.3 | GTGGCCTCCTATGACTACCT | CATCCAACATTCACGCAAGTG | 57.9 | 137 |
HMGCR | NM_000859.2 | CGATGCATAGCCATCCTGTAT | GCTGGAATGACAGCTTCACA | 57.7 | 87 |
HMGCS | NM_001098272.2 | TCTATCCTTCACACAGCTCTTTC | GGCAACAATTCCCACATCTTT | 57.9 | 89 |
HO-1 | NM_002133.2 | CGGCTTCAAGCTGGTGAT | AGCTCTTCTGGGAAGTAGACA | 57.7 | 114 |
HPRT | NM_000194.2 | CACTGGCAAAACAATGCAGACT | GTCTGGCTTATATCCAACACTTCGT | 60.2 | 118 |
IDI | NM_004969.3 | TCTCATTGGGCATGAAGGTC | CATAAAACCTCGGGCTCCTT | 57.6 | 106 |
MFP1 | NM_001966.3 | ATGGATATGGATGGCCAAGG | GCTCCAGTTGGGGAATATCA | 57.1 | 126 |
MFP2 | NM_000414.3 | TGTCGTTGCAGGCCTTATT | CCTCCCAAATCATTCACAACAAC | 57.4 | 148 |
MVD | NM_002461.2 | GGTGGCACCTGTTCTTCTCTCT | CTGATGAGCAGCTGTCTGGAGT | 56.5 | 82 |
MVK | NM_000431.3 | CTGGACACAAGCTTTCTGGA | AAGCCTGCAACCTCCTTTAG | 57.7 | 83 |
PMVK | NM_006556.3 | GCCTTTCGGAAGGACATGAT | GTCACTCACCAGCCAGATG | 58 | 114 |
PPARalpha | NM_005036.4 | CTGGCCAAGAGAATCTACGAG | ACTGGTTCCATGTTGCCAAG | 57.9 | |
PPARbeta | NM_177435.2 | AACATGCAAGGCACTGACTG | CTGCCAAAGTGCTGGGATT | 59 | 129 |
PPARgamma | NM_138712.3 | ATCTTTCAGGGCTGCCAGT | TCGTGGACTCCATATTTGAGG | 58.9 | 131 |
PRDX6 | NM_004905.2 | TTAGTGCCATGTGCCTTTCA | TAGCAACCCACTGCAAGAAG | 57.7 | 144 |
MVD | NM_002461.2 | GGTGGCACCTGTTCTTCTCTCT | CTGATGAGCAGCTGTCTGGAGT | 56.5 | 82 |
MVK | NM_000431.3 | CTGGACACAAGCTTTCTGGA | AAGCCTGCAACCTCCTTTAG | 57.7 | 83 |
PMVK | NM_006556.3 | GCCTTTCGGAAGGACATGAT | GTCACTCACCAGCCAGATG | 58 | 114 |
PPARalpha | NM_005036.4 | CTGGCCAAGAGAATCTACGAG | ACTGGTTCCATGTTGCCAAG | 57.9 | |
PPARbeta | NM_177435.2 | AACATGCAAGGCACTGACTG | CTGCCAAAGTGCTGGGATT | 59 | 129 |
PPARgamma | NM_138712.3 | ATCTTTCAGGGCTGCCAGT | TCGTGGACTCCATATTTGAGG | 58.9 | 131 |
PRDX6 | NM_004905.2 | TTAGTGCCATGTGCCTTTCA | TAGCAACCCACTGCAAGAAG | 57.7 | 144 |
PSP/SPLUNC2 | NM_001319164.1 | GAAGTCTGAGGTGGTGTCAAG | TGCCAAGATTGTCAAGAAGAGA | 58.2 | 107 |
RPL13 | NM_000977. | CGGAATGGCATGGTCTTGA | CCTTACGTCTGCGGATCTTAC | 57.8 | 100 |
SOD1 | NM_000454.4 | AGGATGAAGAGAGGCATGTTG | ATGGTCTCCTGAGAGTGAGAT | 57.7 | 107 |
SOD2 | NM_000636.2 | GTTGGCCAAGGGAGATGTTA | CGTTAGGGCTGAGGTTTGT | 57.5 | 110 |
SQS | NM_001287742.1 | GAAGTCAGTGAGACCAAGAACC | CGCTCTCTGTAGAGCCTTAGA | 58.6 | 76 |
TBP | NM_003194.4 | TGACCCAGCATCACTGTTTC | GCTGGAACTCGTCTCACTATTC | 58.1 | 118 |
Thiolase | NM_001607.3 | GATGCCTTCTTACCCCAACA | CCCAACCACTGCATAAGACC | 57.5 | |
TRX1 | NM_001244938.1 | GGACGCTGCAGGTGATAAA | CACTCTGAAGCAACATCATGAAAG | 57.9 | 102 |
TRX2 | NM_012473.3 | GTTAGAGAAGATGGTGGCCAAG | GCTGACACCTCATACTCAATGG | 58.7 | 99 |
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Meyer, M.T.; Watermann, C.; Dreyer, T.; Wagner, S.; Wittekindt, C.; Klussmann, J.P.; Ergün, S.; Baumgart-Vogt, E.; Karnati, S. Differential Expression of Peroxisomal Proteins in Distinct Types of Parotid Gland Tumors. Int. J. Mol. Sci. 2021, 22, 7872. https://doi.org/10.3390/ijms22157872
Meyer MT, Watermann C, Dreyer T, Wagner S, Wittekindt C, Klussmann JP, Ergün S, Baumgart-Vogt E, Karnati S. Differential Expression of Peroxisomal Proteins in Distinct Types of Parotid Gland Tumors. International Journal of Molecular Sciences. 2021; 22(15):7872. https://doi.org/10.3390/ijms22157872
Chicago/Turabian StyleMeyer, Malin Tordis, Christoph Watermann, Thomas Dreyer, Steffen Wagner, Claus Wittekindt, Jens Peter Klussmann, Süleyman Ergün, Eveline Baumgart-Vogt, and Srikanth Karnati. 2021. "Differential Expression of Peroxisomal Proteins in Distinct Types of Parotid Gland Tumors" International Journal of Molecular Sciences 22, no. 15: 7872. https://doi.org/10.3390/ijms22157872