Apigenin Modulates AnxA6- and TNAP-Mediated Osteoblast Mineralization
Abstract
:1. Introduction
2. Results
2.1. Characterization of the Morphology of Human Fetal Osteoblastic Cell Line (hFOB 1.19 Cells) and Osteosarcoma Cell Line (Saos-2 Cells)
2.2. Effect of Apigenin on hFOB 1.19, Saos-2 and HCASMC Cell Proliferation
2.3. Effect of Apigenin on Mineralization and TNAP Activity of hFOB 1.19 and Saos-2 Cells
2.4. Effect of Apigenin on Protein Profiles of hFOB 1.19 and Saos-2 Cells
2.5. Effect of Apigenin on Protein Distribution in hFOB 1.19 and Saos-2 Cells
2.6. Effect of Apigenin on Vesicles and Minerals Produced by hFOB 1.19 and Saos-2 Cells
2.7. Effect of Apigenin on Protein Distributions in Vesicles Released by hFOB 1.19, Saos-2 and HCASMC Cells
3. Discussion
4. Materials and Methods
4.1. Cell Culture and Treatment
4.2. Morphological Analysis of Cell Culture
4.3. MTT Assay
4.4. Calcium Minerals Detection
4.5. Cell Lysis and TNAP Acivity Assay
4.6. SDS-PAGE and Immunoblot Analysis
4.7. Immunochemistry and Fluorescent Microscopy
4.8. Transmission Electron Microscopy with X-ray Microanalysis (TEM-EDX)
4.9. Transmission Electron Microscopy with Immunogold Labelling (TEM-gold)
4.10. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
AA | ascorbic acid |
ALP | alkaline phosphatase |
AnxA6 | vertebrate annexin A6 |
AR-S | Alizarin Red-S |
β-GP | β-glycerophosphate |
CPC | cetyl pyridinium chloride |
ECM | extracellular matrix |
FBS | fetal bovine serum |
HA | hydroxyapatite |
MVs | matrix vesicles |
PBS | phosphate-buffered saline |
Pi | inorganic phosphate |
pNPP | para-nitro phenyl phosphate |
PPi | inorganic pyrophosphate |
SMC | smooth muscle cells |
TBS | Tris-buffered saline |
TNAP | tissue non-specific alkaline phosphatase |
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Apigenin Concentration (µM) | Relative Co-Localization Area (%) | ||||
---|---|---|---|---|---|
hFOB 1.19 | Saos-2 | ||||
Resting | 0 | 11.34 ± 3.00 | 15.40 ± 3.93 | ||
1 | 19.90 ± 4.23 | 8.48 ± 1.14 | |||
2 | 19.40 ± 2.04 | 7.88 ± 0.67 | |||
5 | 12.53 ± 0.76 | 6.28 ± 2.79 | |||
10 | 10.34 ± 3.53 | 4.59 ± 0.20 | |||
Stimulated | 0 | 13.18 ± 5.92 | 17.93 ± 2.33 | ||
1 | 12.86 ± 5.92 | 8.40 ± 1.07 | |||
2 | 13.96 ± 3.84 | 6.50 ± 0.69 | |||
5 | 8.09 ± 1.77 | 6.11 ± 2.23 | |||
10 | 8.53 ± 3.48 | 5.59 ± 1.94 |
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Mroczek, J.; Pikula, S.; Suski, S.; Weremiejczyk, L.; Biesaga, M.; Strzelecka-Kiliszek, A. Apigenin Modulates AnxA6- and TNAP-Mediated Osteoblast Mineralization. Int. J. Mol. Sci. 2022, 23, 13179. https://doi.org/10.3390/ijms232113179
Mroczek J, Pikula S, Suski S, Weremiejczyk L, Biesaga M, Strzelecka-Kiliszek A. Apigenin Modulates AnxA6- and TNAP-Mediated Osteoblast Mineralization. International Journal of Molecular Sciences. 2022; 23(21):13179. https://doi.org/10.3390/ijms232113179
Chicago/Turabian StyleMroczek, Joanna, Slawomir Pikula, Szymon Suski, Lilianna Weremiejczyk, Magdalena Biesaga, and Agnieszka Strzelecka-Kiliszek. 2022. "Apigenin Modulates AnxA6- and TNAP-Mediated Osteoblast Mineralization" International Journal of Molecular Sciences 23, no. 21: 13179. https://doi.org/10.3390/ijms232113179