FVIII Trafficking Dynamics Across Subcellular Organelles Using CRISPR/Cas9 Specific Gene Knockouts
Abstract
1. Introduction
2. Results
2.1. FVIII Is Localized to ER, ERGIC, Endosomal, and Proteasomal Compartments
2.2. FVIII Co-Localization with Intracellular Markers Places GABARAPKO, CANXKO, and CALRKO Clones in the Same Cluster
2.3. Cell Treatments Align GABARAPKO with CALRKO in FVIII Secretion and Additionally Highlight the Contribution of the Endomembrane System in FVIII Trafficking
2.4. Co-Localization of FVIII with Key Intracellular Landmarks Following Cell Treatments Positions GABARAPKO Close to CANXKO
2.5. Double Knockouts of ER/ERGIC Chaperones and GABARAP Reveal Distinct Effects on FVIII Secretion and Trafficking
3. Discussion
4. Materials and Methods
4.1. Generation of CRISPR/Cas9-Knockouts, Cell Culture and Cell Maintenance
4.2. Genetic Rescue of Double CRISPR/Cas9-Knockouts
4.3. FVIII Activity Measurement
4.4. Measurement of Intracellular ATP Concentration
4.5. Western Blot Analysis
4.6. Immunofluorescence Staining and Microscopy Imaging
4.7. Drug/Chemical and Cell Treatments
4.8. Statistical Analysis and Data Visualization
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
AC | Activity |
AMO1 | Human Bone Marrow Plasmacytoma cells |
ARL8B | ADP-Ribosylation Factor-Like Protein 8B |
ATP | Adenosine Triphosphate |
BFA | Brefeldin A |
BiP | Binding Immunoglobulin Protein |
CALR | Calreticulin |
CANX | Calnexin |
COPI | Coat Protein Complex I |
COPII | Coat Protein Complex II |
CQ | Chloroquine |
CRISPR | Clustered Regularly Interspaced Short Palindromic Repeats |
DKO | Double-KO |
DMSO | Dimethyl Sulfoxide |
ER | Endoplasmic Reticulum |
ERGIC | Endoplasmic Reticulum-Golgi Intermediate Compartment |
ERp57 | Endoplasmic Reticulum Resident Protein 57 |
FAM134B | Family with sequence similarity 134, member B |
GABARAP | Gamma-Aminobutyric Acid Receptor Associated Protein |
GABARAPL1 | GABARAP-Like 1 |
GABARAPL2 | GABARAP-Like 2 |
GAPDH | Glyceraldehyde-3-phosphate dehydrogenase |
GM130 | Golgi Matrix protein 130 |
hiFBS | Heat-Inactivated Fetal Bovine Serum |
ICD | Immunogenic cell death |
KO | Knockout |
LAMPI | lysosome-associated membrane protein I |
LC3B | Microtubule-associated Protein 1 Light Chain 3 B |
LIR | LC3- interacting Region |
LMAN1 | Lectin Mannose binding 1 |
LSEC | Liver Sinusoidal Endothelial Cells |
MCFD2 | Multiple Coagulation Factor Deficiency 2 |
N2a | Neuro-2a cells |
ns | Non significant |
PCA | Principal Component Analysis |
PCC | Pearson’s Correlation Coefficient |
PDI | Protein Disulfide Isomerase |
Rab | Ras-like proteins in brain |
RIPA | Radioimmunoprecipitation Assay |
RT | Room Temperature |
S | Sample |
SD | Standard Deviation |
Sec31a | SEC31 homolog A |
SKO | Single-KO |
SPR | Surface Plasmon Resonance |
TGN46 | Trans-Golgi Network Protein 46 |
Ub | Ubiquitin |
V | Variable |
VAMP | Vesicle-Associated Membrane Protein |
WB | Western Blot |
WT | Wild Type |
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El Hazzouri, S.; Al-Rifai, R.; Surges, N.; Rath, M.; Singer, H.; Oldenburg, J.; El-Maarri, O. FVIII Trafficking Dynamics Across Subcellular Organelles Using CRISPR/Cas9 Specific Gene Knockouts. Int. J. Mol. Sci. 2025, 26, 6349. https://doi.org/10.3390/ijms26136349
El Hazzouri S, Al-Rifai R, Surges N, Rath M, Singer H, Oldenburg J, El-Maarri O. FVIII Trafficking Dynamics Across Subcellular Organelles Using CRISPR/Cas9 Specific Gene Knockouts. International Journal of Molecular Sciences. 2025; 26(13):6349. https://doi.org/10.3390/ijms26136349
Chicago/Turabian StyleEl Hazzouri, Salime, Rawya Al-Rifai, Nicole Surges, Melanie Rath, Heike Singer, Johannes Oldenburg, and Osman El-Maarri. 2025. "FVIII Trafficking Dynamics Across Subcellular Organelles Using CRISPR/Cas9 Specific Gene Knockouts" International Journal of Molecular Sciences 26, no. 13: 6349. https://doi.org/10.3390/ijms26136349
APA StyleEl Hazzouri, S., Al-Rifai, R., Surges, N., Rath, M., Singer, H., Oldenburg, J., & El-Maarri, O. (2025). FVIII Trafficking Dynamics Across Subcellular Organelles Using CRISPR/Cas9 Specific Gene Knockouts. International Journal of Molecular Sciences, 26(13), 6349. https://doi.org/10.3390/ijms26136349