The ER Stress Induced in Human Neuroblastoma Cells Can Be Reverted by Lumacaftor, a CFTR Corrector
Abstract
:1. Introduction
2. Materials and Methods
2.1. Reagents
2.2. Cell Culture
2.3. Experimental Protocol
2.4. Cell Viability
2.5. Protein Extraction and Western Blotting Assay
2.6. RNA Extraction and Real-Time RT-PCR Protocol
2.7. Calcium Signaling Assay
2.8. Intracellular and Mitochondrial ROS Detection
2.9. Caspase 4 Measurement
2.10. Hypodiploid DNA Detection
2.11. Analytical Statistics
3. Results
3.1. Role of Vx-809 in UPR Pathway
3.2. “Corrector” Vx-809 Interferes on Calcium Signaling
3.3. Vx-809 Counteract Thapsigargin-Induced Oxidative Stress
3.4. Vx-809 Interferes with the Apoptotic Pathway
4. Discussion
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
AD | Alzheimer’s disease |
ER | Endoplasmic reticulum |
UPR | Unfolded protein response |
IRE1α | Inositol-requiring enzyme 1 α |
PERK | Protein kinase RNA-like ER kinase |
ATF6 | Activating transcription Factor 6 |
GRP78/BiP | Glucose-regulated protein 78/binding immunoglobulin protein |
eIF2α | Eukaryotic translation initiation factor 2A |
ATF4 | Activating transcription factor 4 |
CHOP | C/EBP homologous protein |
ERAD | ER-associated degradation |
Vx-809 | Lumacaftor |
CF | Cystic fibrosis |
NBD1 | Nucleotide-binding domain 1 |
TMD1 | Transmembrane domain 1 |
TG | Thapsygargin |
NDs | Neurodegenerative diseases |
ROS | Reactive oxygen species |
PD | Parkinson’s disease |
PrD | Prion disease |
Xbp1 | X-box binding protein 1 |
Xbp1s | Spliced Xbp1 |
CFTR | Cystic fibrosis transmembrane conductance regulator |
SODIII | Superoxide dismutase III |
DMEM | Dulbecco’s modified Eagle’s Medium |
FBS | Fetal bovine serum |
PBS | Phosphate-buffered saline |
MTT | 3-[4,5-dimetiltiazol]-2,5-diphenyl-2H-tetrazolium bromide |
Sp | Staurosporine |
ECL | Enhanced chemiluminescence |
HBSS | Hank’s balanced salt solution |
H2DCF-DA | 2′,7′-dichlorofluorescin diacetate |
MitoSOX | Mitochondrial superoxide |
PI | Propidium iodide |
SERCA | Sarco/endoplasmic reticulum Ca2+-ATPase |
ALSIDP | Amyotrophic lateral sclerosisIntrinsically disordered proteins |
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Pecoraro, M.; Serra, A.; Pascale, M.; Franceschelli, S. The ER Stress Induced in Human Neuroblastoma Cells Can Be Reverted by Lumacaftor, a CFTR Corrector. Curr. Issues Mol. Biol. 2024, 46, 9342-9358. https://doi.org/10.3390/cimb46090553
Pecoraro M, Serra A, Pascale M, Franceschelli S. The ER Stress Induced in Human Neuroblastoma Cells Can Be Reverted by Lumacaftor, a CFTR Corrector. Current Issues in Molecular Biology. 2024; 46(9):9342-9358. https://doi.org/10.3390/cimb46090553
Chicago/Turabian StylePecoraro, Michela, Adele Serra, Maria Pascale, and Silvia Franceschelli. 2024. "The ER Stress Induced in Human Neuroblastoma Cells Can Be Reverted by Lumacaftor, a CFTR Corrector" Current Issues in Molecular Biology 46, no. 9: 9342-9358. https://doi.org/10.3390/cimb46090553