Xylosyltransferase-Deficiency in Human Dermal Fibroblasts Induces Compensatory Myofibroblast Differentiation and Long-Term ECM Reduction
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Culture
2.2. CRISPR/Cas9-Based KO in Human Dermal Fibroblasts
2.3. Fluorescence-Based Cell Sorting and Cell Separation
2.4. TA Cloning
2.5. siRNA-Based Double-Knockdown (dKD)
2.6. RNA Extraction, Reverse Transcription and Quantitative Real-Time Polymerase Chain Reaction (qPCR)
2.7. Xyloslytransferase Activity Assay
2.8. Galactosyltransferase Activity Assay
2.9. Determination of the Sulfated GAG (sGAG) Concentration in Fibroblasts
2.10. Western Blot
2.11. Bicinchoninic Acid Assay
2.12. Determination of the Pro-MMP1 Concentration in the Cell Culture Supernatants
2.13. Determination of the TGFβ1 Concentration in Cell Culture Supernatants
2.14. Determination of Cell Viability and Caspase Activity in Fibroblasts
2.15. Quantitative Senescence Assay
2.16. Determination of the Interleukin 6 (IL6) Concentration in Cell Culture Supernatants
2.17. Determination of the Intracellular Ca2+ Concentration of Fibroblasts
2.18. Inhibition of the TGFβ Receptor Kinase and Focal Adhesion-Associated Kinase (FAK) in Fibroblasts
2.19. Immunofluorescence
2.20. Statistical Analysis
3. Results
3.1. Generation of Separated CRISPR/Cas9-Mediated XYLT1 and XYLT2 KOs in Human Dermal Fibroblasts and Their Initial Characterization
3.2. Establishment of a Simultaneous XYLT1 and XYLT2 KO in Dermal Fibroblasts
3.3. Establishment of a Simultaneous XYLT1 and XYLT2 dKD in Dermal Fibroblasts
3.4. Analysis of TGFβ Signal Transduction following XYLT1/XYLT2 dKD in Dermal Fibroblasts
4. Discussion
4.1. Characterization of the Introduced Genetic Mutations
4.2. Analysis of Tetrasaccharide Linker Transferase Expressions and Their Activities
4.3. Analysis of sGAG Concentration and DCN and ACAN mRNA Expressions
4.4. Analysis of the Expression of ECM-Associated Proteins
4.5. Analysis of Viability, Senescence, Apoptosis and Oxidative Stress
4.6. Characterization of XYLT1- and XYLT2-Double-Deficient Dermal Fibroblasts
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Target | 5′–3′ Sequence | IDT Label |
---|---|---|
XYLT1 | CTTACTGCCGCCACAAGTTA | XYLT1.1.AA |
XYLT2 | GGTTACTGCCCGTACCACCT | XYLT2.1.AB |
Target | 5′–3′ Sequence |
---|---|
XYLT1 | GCAUCAUGCUACCAAUCUGtt ttCGUAGUACGAUGGUUAGAC |
XYLT2 | GGCCGUUUAUCACGAGCAGtt ttCCGGCAAUAGUGCUCGUC |
Target | Sequence | TA/°C | Product Size/bp |
---|---|---|---|
ACAN | CACCCCATGCAATTTGAG GCCACTGTGCCCTTTTTA | 63 | 158 |
ACTA2 | GACCGAATGCAGAAGGAG CGGTGGACAATGGAAGG | 59 | 169 |
B2M | TGTGCTCGCGCTACTCTCTCTT CGGATGGATGAAACCCAGACA | 63 | 137 |
B3GALT6 | CCCCGCTGTGGTCTTTGTTG CGCCCCCGTTTCTTCCTC | 63 | 188 |
B3GAT3 | GCTGTTTGAGGAGATGCGCTG TCAGAAGACTGCTCTCCAGGT | 63 | 251 |
B4GALT7 | GCCATGCACAGTGATCAGAG CCCTACACTGTGTCTCTGCA | 63 | 196 |
COL1A1 | GATGTGCCACTCTGACT GGGTTCTTGCTGATG | 63 | 151 |
DCN | CCTTCCGCTGTCAATG GCAGGTCTAGCAGAGTTG | 63 | 102 |
GAPDH | AGGTCGGAGTCAACGGAT TCCTGGAAGATGGTGATG | 59 | 223 |
IL1B | ACAGATGAAGTGCTCCTTCCA GTCGGAGATTCGTAGCTGGAT | 63 | 73 |
IL6 | ACAGCCACTCACCTCTTCAG GTGCCTCTTTGCTGCTTTCAC | 63 | 122 |
IL8 | GAACTGAGAGTGATTGAGAGTGGA CTCTTCAAAAACTTCTCCACAACC | 63 | 125 |
MMP1 | AGAAACACAAGAGCAAGATGTG TGGCGTGTAATTTTCAATCCTGT | 63 | 298 |
NOX4 | CTTCCGTTGGTTTGCAGATT GAATTGGGTCCACAACAGA | 63 | 246 |
RPL13A | CGGAAGGTGGTGGTCGTA CTCGGGAAGGGTTGGTGT | 63 | 115 |
SDHA | AACTCGCTCTTGGACCTG GAGTCGCAGTTCCGATGT | 63 | 177 |
TGFB1 | GCGATACCTCAGCAACC ACGCAGCAGTTCTTCTCC | 63 | 331 |
XYLT1 | GAAGCCGTGGTGAATCAG CGGTCAGCAAGGAAGTAG | 63 | 281 |
XYLT2 | ACACAGATGACCCGCTTGTGG TTGGTGACCCGCAGGTTGTTG | 63 | 139 |
Primary Antibody | Dilution | Secondary Antibody | Dilution |
---|---|---|---|
Rabbit-anti-COL1A1 (ab34710; Abcam, Cambridge, UK) | 1:10,000 | Goat-anti-rabbit IgG-HRP polyclonal (ab150113; Abcam, Cambridge, UK) | 1:5000 |
Mouse-anti-αSMA (GA61161-2; Cell Signaling Technology, Cambridge, UK) | 1:1000 | Horse-anti-mouse igG-HRP polyclonal (ab150078; Abcam, Cambridge, UK) | 1:2000 |
Mouse-anti-gapdh (ab8245; Abcam, Cambridge, UK) | 1:5000 | Horse-anti-mouse igG-HRP polyclonal (ab150078; Abcam, Cambridge, UK) | 1:2000 |
Primary Antibody | Dilution | Secondary Antibody | Dilution |
---|---|---|---|
Rabbit-anti-COL1A1 (ab34710; Abcam, Cambridge, UK) | 1:100 | Goat-anti-rabbit (Alexa-555) (A-32732; Thermo Fisher Scientific, USA) | 1:200 |
Mouse-anti-αSMA (GA61161-2; Cell Signaling Technology, Cambridge, UK) | 1:50 | Goat-anti-mouse (Alexa-488) (A-11001; Thermo Fisher Scientific, USA) | 1:200 |
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Share and Cite
Kleine, A.; Kühle, M.; Ly, T.-D.; Schmidt, V.; Faust-Hinse, I.; Knabbe, C.; Fischer, B. Xylosyltransferase-Deficiency in Human Dermal Fibroblasts Induces Compensatory Myofibroblast Differentiation and Long-Term ECM Reduction. Biomedicines 2024, 12, 572. https://doi.org/10.3390/biomedicines12030572
Kleine A, Kühle M, Ly T-D, Schmidt V, Faust-Hinse I, Knabbe C, Fischer B. Xylosyltransferase-Deficiency in Human Dermal Fibroblasts Induces Compensatory Myofibroblast Differentiation and Long-Term ECM Reduction. Biomedicines. 2024; 12(3):572. https://doi.org/10.3390/biomedicines12030572
Chicago/Turabian StyleKleine, Anika, Matthias Kühle, Thanh-Diep Ly, Vanessa Schmidt, Isabel Faust-Hinse, Cornelius Knabbe, and Bastian Fischer. 2024. "Xylosyltransferase-Deficiency in Human Dermal Fibroblasts Induces Compensatory Myofibroblast Differentiation and Long-Term ECM Reduction" Biomedicines 12, no. 3: 572. https://doi.org/10.3390/biomedicines12030572
APA StyleKleine, A., Kühle, M., Ly, T.-D., Schmidt, V., Faust-Hinse, I., Knabbe, C., & Fischer, B. (2024). Xylosyltransferase-Deficiency in Human Dermal Fibroblasts Induces Compensatory Myofibroblast Differentiation and Long-Term ECM Reduction. Biomedicines, 12(3), 572. https://doi.org/10.3390/biomedicines12030572