Increased H3K9me3 and F-Actin Reorganization in the Rapid Adaptive Response to Hypergravity in Human T Lymphocytes
Abstract
:1. Introduction
2. Results
2.1. Hypergravity Did Not Compromise Cell Viability
2.2. Hypergravity Only Minimally Affected H3K4me3 Histone Modification
2.3. Dynamic Response of H3K9me3 Histone Modification in Hypergravity Conditions
2.4. Quantitative Analysis of Cytoskeletal Changes in Hypergravity
2.5. Dynamic Changes of Nuclear Morphology in Hypergravity
2.6. Characterization of Histone Modification Spots in Hypergravity
2.7. Spatial Distribution of Histone Modification Spots in Relation to the Nuclear Periphery in Hypergravity
2.8. Dynamic Response of H3K9me3 Histone Modification Spots to Hypergravity
3. Discussion
4. Materials and Methods
4.1. Cell Culture
4.2. Centrifugation Experiment Design
4.3. Immunocytochemical Staining
4.4. Widefield Microscopy
4.5. Confocal Microscopy
4.6. Image Analysis
4.7. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
LINC | Linker of nucleoskeleton to cytoskeleton |
RFI | Relative fluorescence intensity |
SEM | Standard error of mean |
CCC | Cell culture control |
HWC | Hardware control |
NC | Negative control |
PC | Positive control |
H3K4me3 | Histone 3 lysine 4 trimethylation |
H3K9me3 | Histone 3 lysine 9 trimethylation |
H3K4ac | Histone 3 lysine 4 acetylation |
H3K27me3 | Histone 3 lysine 27 trimethylation |
HM | Histone modification |
TUNEL | Terminal deoxynucleotidyl transferase dUTP nick end labeling |
COMPASS | Complex Proteins Associated with Set1 |
HMT | Histone methyltransferase |
LAD | Lamin associated domain |
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ID | Group | Gravitational Force (× g) | Centrifuge Time (Tc) | Incubation Time (Ti) |
---|---|---|---|---|
CCC | Cell culture control (CCC) | 1 | 0 | 0 |
HWC | Hardware control (HWC) | 1 | 0 | 15 min |
1.8g, 20s | Hypergravity (parabolic flight simulation) | 1.8 | 20 s | 14 min 40 s |
9g, 20s | Hypergravity | 9 | 20 s | 14 min 40 s |
9g, 75s | Hypergravity (sounding rocket simulation) | 9 | 75 s | 13 min 45 s |
9g, 15m | Hypergravity | 9 | 15 min | 0 |
9g, 1h | Hypergravity | 9 | 60 min | 0 |
Analyses | Epitope/Parameter | CCC | HWC | 1.8g20s | 9g20s | 9g75s | 9g15m | 9g1h | |
---|---|---|---|---|---|---|---|---|---|
Widefield Overall RFI | H3K4me3 | 0.80 | 1.00 | 0.74 | 0.92 | 0.86 | 0.97 | 0.89 | |
H3K9me3 | 1.00 | 1.00 | 1.63 | 1.69 | 1.90 | 2.18 | 0.95 | ||
F-Actin | 1.01 | 0.94 | 0.68 | 0.59 | 0.70 | 0.66 | 1.22 | ||
Nuclear Shape | Width (µm) | 5.20 | 5.40 | 5.56 | 5.62 | 5.77 | 5.82 | 5.96 | |
Height (µm) | 3.62 | 3.44 | 3.21 | 3.15 | 3.03 | 3.07 | 2.98 | ||
Oblate | 0.36 | 0.39 | 0.43 | 0.46 | 0.47 | 0.45 | 0.46 | ||
Sphericity | 0.92 | 0.92 | 0.91 | 0.90 | 0.89 | 0.90 | 0.90 | ||
HM Spot Number | H3K4me3 | 1404 | 1617 | 681 | 1162 | 897 | 778 | 1454 | |
H3K9me3 | 576 | 533 | 561 | 814 | 735 | 957 | 437 | ||
HM Spot Peripheral Distance | H3K4me3 | Skew | −0.69 | −0.77 | −0.72 | −0.66 | −0.65 | −0.73 | −0.52 |
Mean (µm) | −1.25 | −1.20 | −1.24 | −1.25 | −1.18 | −1.20 | −1.44 | ||
H3K9me3 | Skew | −0.88 | −1.00 | −0.98 | −0.69 | −0.95 | −0.78 | −0.62 | |
Mean (µm) | −1.09 | −1.17 | −1.18 | −1.08 | −1.14 | −1.12 | −1.22 | ||
HM Spot RFI | H3K4me3 | Skew | 0.25 | 0.21 | 0.66 | 0.18 | 0.12 | 0.33 | 0.31 |
Mean | 1.07 | 1.00 | 1.07 | 1.30 | 1.20 | 1.06 | 0.95 | ||
H3K9me3 | Skew | 1.08 | 1.08 | 0.88 | 0.95 | 1.08 | 0.89 | 1.22 | |
Mean | 1.04 | 1.00 | 1.11 | 1.08 | 1.07 | 1.16 | 0.92 |
Epitope | Excitation (nm) | Emission (nm) | Exposure Time (ms) | Laser Power | Gain |
---|---|---|---|---|---|
DAPI | 390 | 460–480 | 100 | 60% | low noise & high well |
H3K4me3/H3K9me3 (Alexa 488) | 475 | 535–570 | 200 | 60% | low noise & high well |
Phalloidin (Alexa 647) | 635 | >695 | 300 | 60% | low noise & high well |
TUNEL | 635 | >695 | 200 | 40% | low noise & high well |
Epitope | Excitation (nm) | Emission (nm) | Exposure Time (ms) | Laser Power | Gain |
---|---|---|---|---|---|
DAPI | 405 | 425–506 | 100 | 30% | 2 |
H3K4me3/H3K9me3 (Alexa 488) | 488 | 506–620 | 200 | 50% | 2 |
Phalloidin (Alexa 647) | 647 | 650–776 | 300 | 50% | 2 |
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Wernlé, K.; Thiel, C.S.; Ullrich, O. Increased H3K9me3 and F-Actin Reorganization in the Rapid Adaptive Response to Hypergravity in Human T Lymphocytes. Int. J. Mol. Sci. 2023, 24, 17232. https://doi.org/10.3390/ijms242417232
Wernlé K, Thiel CS, Ullrich O. Increased H3K9me3 and F-Actin Reorganization in the Rapid Adaptive Response to Hypergravity in Human T Lymphocytes. International Journal of Molecular Sciences. 2023; 24(24):17232. https://doi.org/10.3390/ijms242417232
Chicago/Turabian StyleWernlé, Kendra, Cora S. Thiel, and Oliver Ullrich. 2023. "Increased H3K9me3 and F-Actin Reorganization in the Rapid Adaptive Response to Hypergravity in Human T Lymphocytes" International Journal of Molecular Sciences 24, no. 24: 17232. https://doi.org/10.3390/ijms242417232