Overexpression of GRP78/BiP in P-Glycoprotein-Positive L1210 Cells is Responsible for Altered Response of Cells to Tunicamycin as a Stressor of the Endoplasmic Reticulum
Abstract
1. Introduction
2. Materials and Methods
2.1. Cells, Culture Conditions, and Transfections
2.2. Effect of Culturing in Media Containing Tunicamycin on the Viability of S, R and T Cells
2.3. Detection of Tunicamycin-Induced Apoptosis and Necrosis in S, R, and T Cells
2.4. Monitoring cell cycle progression
2.5. Western Blotting
2.6. Determination of ER Stress-Induced Factors and Chaperones by RT-PCR
2.7. Determination of Stress Response Proteins by qPCR
2.8. Visualization of GRP78/BiP in S, R, and T Cells Using Immunofluorescence Confocal Microscopy
2.9. Detection of GRP78/Bip on Cell Surface of L1210 cells by Flow Cytometry
3. Results
3.1. Effect of Tunicamycin on S, R, and T Cell Cycle Progression
3.2. Endoplasmic Stress Receptors and Activation of the UPR
3.3. Downregulation of CHOP Expression After Transfection of S Cells With GRP78/BiP
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
ABC | ATP-binding cassette |
ALL | acute lymphoblasticleukemia |
calcein AM | calcein acetoxymethyl |
AML | Acute myeloid leukemia |
ATF4 | activatingtranscription factor 4 |
ATF6 | activating transcription factor 6 |
ATF6/f | 50-kDa proteolytic fragment of ATF6 |
Bax | Bcl-2-associated X protein |
Bcl-2 | B-cell lymphoma 2 protein |
BSA | Bovine serum albumin |
cAMP | Cyclic adenosine monophosphate |
CDK4 | Cyclindependentkinase 4 |
CHOP | C/EBP homologous protein |
CNX | Calnexin |
CRT | calreticulin |
Ct value | cycle threshold value |
DAPI | 4′-6-diamidino-2-phenylindole |
DMBA | dimethylbenzanthracene |
DSMZ | German Collection of Microorganisms and Cell Cultures |
eIF2 | eukaryotic translation-initiation factor 2 |
ER | endoplasmic reticulum |
ERAD | Endoplasmic-reticulum-associated protein degradation |
FAV | Fluorescein linked annexin V |
FP | folded protein |
FITC | Fluorescein isothiocyanate |
GAPDH | Glyceraldehyde 3-phosphate dehydrogenase |
GRP78/BiP | glucose-regulated protein 78/Binding immunoglobulin protein |
GRP94 | glucose-regulated protein 94 |
SIRE1 | inositol-requiring enzyme 1 |
MDR | multidrug resistance |
MTT | 3-(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide |
PBS | Phosphate-buffered saline |
PCR | polymerasechain reaction |
PERK | pancreatic ER kinase-like ER kinase |
P-gp | P-glycoprotein |
PI | propidium iodide |
SDS-PAGE | sodium dodecyl sulfate–polyacrylamide gel electrophoresis |
UPR | unfolded proteinresponse |
UP | unfolded protein |
UV | ultraviolet |
s/us/t XBP1 | spliced/unspliced/total X-boxbinding protein 1 |
qRT-PCR | quantitative RT-PCR |
RPMI 1640 | Roswell Park MemorialInstitute 1640 medium |
HRP | horseradish peroxidase |
VCR | vincristine |
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Gene | Forward Primer | Revers Primer | bp |
---|---|---|---|
GAPDH b | 5′-TAT GTC GTG GAG TCT ACT GGT GTC-3′ | 5′-GTC ATC ATA CTT GGC AGG TTT CTC-3′ | 492 |
IRE1α b | 5′-AAC ACA CCG ACC ACC GTA TC-3′ | 5′-AGG GTC CTG GGT AAG GTC TC-3′ | 282 |
PERK b | 5′-GCC GAC GAT CAA ATG GAA GC-3′ | 5′-GTG GGG CTG AGG ATG GAA AA-3′ | 370 |
ATF6 b | 5′-TGG AAG TGG GAA GAT CGG GA-3′ | 5′-AGC CAC AGG TCC TCT TTA GG-3′ | 312 |
XBP1 a | 5′-GAA CCA GGA GTT AAG AAC ACG-3′ | 5′-AGG CAA CAG TGT CAG AGT CC-3′ | us 205 s 179 |
CHOP b | 5′-GGA ACC TGA GGA GAG GTG TTC-3′ | 5′-TGC AGA TCC TCA TAC CAG G-3′ | 162 |
GRP94 b | 5′-GGG GAG GTC ACC TTC AAG TC-3′ | 5′-TGA GGG GGA GAT CAT CGG AA-3′ | 199 |
Cnx b | 5′-AGT GGG AAG TAG ATG AGA TGA AGG-3′ | 5′-ATA CAC CTG TCT TGG GAT TTT TGT-3′ | 333 |
cyclin D1 b | 5′-TCA CCC TGA GAG TAG GGA GC-3′ | 5′-GGC CTT CAG GCA AAA ACC AG-3′ | 592 |
cyclin A b | 5′-AGC AGA ACT CAT TCG GCT CT-3′ | 5′-CAA GGG AAA AGG AAG AAG AAG AGA A-3′ | 297 |
cyclin E b | 5′-ATG TTA CAG ATG GCG CTT GC-3′ | 5′-GAG GAC ACC ATA AGG AAA TCT GA-3′ | 254 |
cyclin B1 b | 5′-CAG TTG TGT GCC CAA GAA GA-3′ | 5′-CTA CGG AGG AAG TGC AGA GG-3′ | 216 |
Bcl-2 b | 5′-GGC TGG GGA TGA CTT CTC TC-3′ | 5′-GCA TGC TGG GGC CAT ATA GTT-3‘ | 323 |
Bax b | 5′-ATC CAA GAC CAG GGT GGC T-3′ | 5′-CTT CCC CCA TTC ATC CCA GG-3′ | 197 |
Gene | Forward Primer | Revers Primer |
---|---|---|
sXBP1 c,s | 5′-GTC CAT GGG AAG ATG TTC TGG-3′ | 5′-CTG AGT CCG AAT CAG GTG CAG-3′ |
usXBP1 c, us | 5′-GTC CAT GGG AAG ATG TTC TGG-3′ | 5′-CAG CAC TCA GAC TAT GTG CA-3′ |
totalXBP1 c,t | 5′-GTC CAT GGG AAG ATG TTC TGG-3′ | 5′-TGG CCG GGT CTG CTG AGT CCG-3′ |
CHOP c | 5′-AGG TGA AAG GCA GGG ACT CA-3′ | 5′-CCA CCA CAC CTG AAA GCA GAA-3′ |
GRP78/BiP c | 5′-TTT TCT GAT GTA TCC TCT TCA CCA GT-3′ | 5′-TTC AGC CAA TTA TCA GCA AAC TCT-3′ |
GRP94 c | 5′-CAA ATG GAG AAG ATT CCG CC-3′ | 5′-AAG AAT GAA GGA AAA ACA GGA CAA AA-3′ |
β-actin c | 5′-TGT CCA CCT TCC AGC AGA T-3′ | 5′-AGC TCA GTA ACA GTC CGC C-3′ |
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Share and Cite
Šereš, M.; Pavlíková, L.; Boháčová, V.; Kyca, T.; Borovská, I.; Lakatoš, B.; Breier, A.; Sulová, Z. Overexpression of GRP78/BiP in P-Glycoprotein-Positive L1210 Cells is Responsible for Altered Response of Cells to Tunicamycin as a Stressor of the Endoplasmic Reticulum. Cells 2020, 9, 890. https://doi.org/10.3390/cells9040890
Šereš M, Pavlíková L, Boháčová V, Kyca T, Borovská I, Lakatoš B, Breier A, Sulová Z. Overexpression of GRP78/BiP in P-Glycoprotein-Positive L1210 Cells is Responsible for Altered Response of Cells to Tunicamycin as a Stressor of the Endoplasmic Reticulum. Cells. 2020; 9(4):890. https://doi.org/10.3390/cells9040890
Chicago/Turabian StyleŠereš, Mário, Lucia Pavlíková, Viera Boháčová, Tomáš Kyca, Ivana Borovská, Boris Lakatoš, Albert Breier, and Zdena Sulová. 2020. "Overexpression of GRP78/BiP in P-Glycoprotein-Positive L1210 Cells is Responsible for Altered Response of Cells to Tunicamycin as a Stressor of the Endoplasmic Reticulum" Cells 9, no. 4: 890. https://doi.org/10.3390/cells9040890
APA StyleŠereš, M., Pavlíková, L., Boháčová, V., Kyca, T., Borovská, I., Lakatoš, B., Breier, A., & Sulová, Z. (2020). Overexpression of GRP78/BiP in P-Glycoprotein-Positive L1210 Cells is Responsible for Altered Response of Cells to Tunicamycin as a Stressor of the Endoplasmic Reticulum. Cells, 9(4), 890. https://doi.org/10.3390/cells9040890