Hypomyelinating Leukodystrophy 7 (HLD7)-Associated Mutation of POLR3A Is Related to Defective Oligodendroglial Cell Differentiation, Which Is Ameliorated by Ibuprofen
Abstract
:1. Introduction
2. Material and Methods
2.1. Primary and Secondary Antibodies, Inhibitors, and Plasmid Constructions
2.2. Cell Culture and Differentiation
2.3. Transfection into Cells
2.4. Confocal Microscopic Images
2.5. Polyacrylamide Gel Electrophoresis and Immunoblotting
2.6. Statistical Analysis
2.7. Ethics Statement
3. Results
3.1. The R140X Mutant Proteins of POLR3A Are Aggregated and Present in Lysosomes in FBD−102b Cells
3.2. Morphological Differentiation Is Inhibited in Cells Harboring the R140X Mutant Constructs of POLR3A
3.3. Ibuprofen Reverses Phenotypes in Cells Harboring the R140X Mutant Constructs of POLR3A
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Product Name | Target or Description | Company | Cat. No. | Lot. No. | Final Conc. for Antibody |
---|---|---|---|---|---|
Anti-eIF4EBP1 (phospho T37) antibody | A phospho-peptide corresponding to residues surrounding theronine 37 of eIF4EBP1 | abcam | ab75767 | GR88680-14 | IF, 1/100 |
Anti-F-actin antibody | Filamentous actin (F-actin) | abcam | ab205 | GR319251-7 | IB, 1/80,000 |
Anti-GFP mAb | Green fluorescent protein (GFP) | MBL | 598 | “078” | IB, 1/80,000 |
Anti-Halo Tag pAb | Halo Tag | Promega | G9281 | “0000028445” | IB, 1/20,000 |
HaloTag Oregon Green Ligand | Halo-tag ligand | Promega | G2802 | “0000391849” | IF, 1/1000 |
Anti-KDEL mAb | KDEL-containing peptide of the endoplasmic reticulum (ER)-resident glucose-regulated protein (GRP78) | MBL | M181-3 | “004” | IF, 1/500 |
Anti-PLP1 antibody | Myelin proteolipid protein 1 (PLP1) | Atlas Antibodies | HPA004128 | B115828 | IB, 1/500 |
Anti-RPS6 (phospho S240 + S244) antibody | Synthetic peptide within human S6 protein to the C-terminus (phospho S240 + S244) | abcam | ab215214 | GR3205097-3 | IF, 1/100 |
CNPase (D83E10) XP Rabbit mAb | 2’, 3’-cyclic nucleotide 3’-phospho-diesterase (CNPase) | Cell Signaling Technology | #5664 | 1 | IB, 1/500 |
Anti-LAMP-1 antibody (H4A3) | Lysosomal-associated membrane protein1 (LAMP1) | Santa Cruz Biotechnology | sc-20011 | J0919 | IF, 1/200 |
Purified Mouse Anti-GM130 antibody | Golgi matrix protein of 130 kDa (GM130) | BD Biosciences | 610823 | 8352796 | IF, 1/500 |
Anti-Sox-10 antibody (A-2) | SRY-related HMG-box 10 (Sox10) | Santa Cruz Biotechnology | sc-365692 | J0720 | IB, 1/500 |
Anti-LC3 protein | Microtubule-associated protein 1 light chain 3 (LC3) | MBL | M152-3 | “057” | IF, 1/500 |
Anti-mTOR antibody (A30) | Mechanistic target of rapamycin (mTOR [kinase ]) | Santa Cruz Biotechnology | sc-517464 | H2621 | IB, 1/100 |
Alexa Fluor 488 goat anti-mouse IgG (H+L) | Mouse IgG (H+L) conjugated with Alexa Fluor 488 | Thermo Fisher Scientific | A-11001 | 774904 | IF, 1/500 |
Alexa Fluor 488 goat anti-rabbit IgG (H+L) | Rabbit IgG (H+L) conjugated with Alexa Fluor 488 | Thermo Fisher Scientific | A-11008 | 751094 | IF, 1/500 |
Alexa Fluor 594 goat anti-mouse IgG (H+L) | Mouse IgG (H+L) conjugated with Alexa Fluor 594 | Thermo Fisher Scientific | A-11005 | 2043369 | IF, 1/500 |
Alexa Fluor 594 goat anti-rabbit IgG (H+L) | Rabbit IgG (H+L) conjugated with Alexa Fluor 594 | Thermo Fisher Scientific | A-11012 | 2018240 | IF, 1/500 |
Anti-IgG (H+L chain) (Mouse) pAb-HRP | Mouse IgG F(ab’) conjugated with horseradish peroxidase | MBL | 330 | 366 | IB, 1/5000 |
Anti-IgG (H+L chain) (Mouse) pAb-HRP | Mouse IgG F(ab’) conjugated with horseradish peroxidase | MBL | 330 | 366 | IB, 1/5000 |
AH6809 | Prostaglandin receptor antagonist | Med Chem Express | HY-10418 | 27839 |
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Sawaguchi, S.; Tago, K.; Oizumi, H.; Ohbuchi, K.; Yamamoto, M.; Mizoguchi, K.; Miyamoto, Y.; Yamauchi, J. Hypomyelinating Leukodystrophy 7 (HLD7)-Associated Mutation of POLR3A Is Related to Defective Oligodendroglial Cell Differentiation, Which Is Ameliorated by Ibuprofen. Neurol. Int. 2022, 14, 11-33. https://doi.org/10.3390/neurolint14010002
Sawaguchi S, Tago K, Oizumi H, Ohbuchi K, Yamamoto M, Mizoguchi K, Miyamoto Y, Yamauchi J. Hypomyelinating Leukodystrophy 7 (HLD7)-Associated Mutation of POLR3A Is Related to Defective Oligodendroglial Cell Differentiation, Which Is Ameliorated by Ibuprofen. Neurology International. 2022; 14(1):11-33. https://doi.org/10.3390/neurolint14010002
Chicago/Turabian StyleSawaguchi, Sui, Kenji Tago, Hiroaki Oizumi, Katsuya Ohbuchi, Masahiro Yamamoto, Kazushige Mizoguchi, Yuki Miyamoto, and Junji Yamauchi. 2022. "Hypomyelinating Leukodystrophy 7 (HLD7)-Associated Mutation of POLR3A Is Related to Defective Oligodendroglial Cell Differentiation, Which Is Ameliorated by Ibuprofen" Neurology International 14, no. 1: 11-33. https://doi.org/10.3390/neurolint14010002