Curcumin Analogue C1 Promotes Hex and Gal Recruitment to the Plasma Membrane via mTORC1-Independent TFEB Activation
Abstract
:1. Introduction
2. Results
2.1. Jurkat Cell Stimulation Promotes TFEB Nuclear Translocation and Induces Cell Exocytosis
2.2. External Leaflet Microdomain-Associated Hex and Gal Increase after Jurkat Cell Stimulation
2.3. TFEB Nuclear Translocation Induced by the Curcumin Analogue C1 Increases both the Expression of HEXB, HEXA, and GLB1 genes and the Recruitment of Glycohydrolases to Lipid Microdomains
2.4. Curcumin Analogue C1 Promotes TFEB Activation without Inhibiting mTORC1 Activity
2.5. Curcumin Analogue C1 Promotes an Increase in both Expression and Activity of Hex and Gal and Their Recruitment on the Cell Surface of SH-SY5Y Cells
3. Discussion
4. Materials and Methods
4.1. C1 Synthesis
4.2. Cell Culturing
4.3. Drugs and Cell Treatments
4.4. TFEB RNA Interference
4.5. Cytosolic, Nuclear, and Enriched Plasma Membrane Extracts
4.6. Isolation of Lipid Microdomains
4.7. Quantitative PCR
4.8. Horseradish Peroxidase Assay
4.9. Isolation of Cell Surface Lipid Microdomain Proteins
4.10. Immunoblotting and Dot Blot Analysis
4.11. Determination of Enzyme Activities
4.12. Immunofluorescence Analysis
4.13. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
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Target Gene (Human) | Sequence (5′→3′) | |
---|---|---|
HEXB | Sense | TTTGGGAGGAGATGAAGTGG |
Antisense | AAACCTCCTGCCAGACAATG | |
HEXA | Sense | GCATTTGAAGGTACCCCTGA |
Antisense | TCAACTTGTTGCTCCACAGC | |
GLB1 | Sense | GTTATAACAGTGCAGGTTGAAAATGAA |
Antisense | CCCAGATGGTGGCGAAAG | |
TFEB | Sense | TCTGCAGCAGTCGCAGCAT |
Antisense | CCAATGTGCAGCATGGCCA | |
ACTB | Sense | AGAAAATCTGGCACCACACC |
Antisense | GGGGTGTTGAAGGTCTCAAA |
Target | Type | Producer (Cat. Number) | Working Dilution |
---|---|---|---|
TFEB | Goat IgG | Abcam (ab2636) | 1:1000 |
H3 | Rabbit IgG | Abcam (ab1791) | 1:5000 |
β-actin | Mouse IgG2a | Sigma-Aldrich (A5316) | 1:5000 |
LC3B | Rabbit IgG | Cell Signaling Technology (#2775) | 1:1000 |
flotillin-2 | Mouse IgG1 | BD Biosciences (610383) | 1:5000 |
Lck | Mouse IgG2b | Santa Cruz Biotechnology (sc-433) | 1:200 |
Phospho-S6 ribosomal protein (S235/236) | Rabbit IgG | Cell Signaling Technology (#2211) | 1:1000 |
S6 ribosomal protein | Rabbit IgG | Cell Signaling Technology (#2217) | 1:1000 |
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Magini, A.; Polchi, A.; Di Meo, D.; Buratta, S.; Chiaradia, E.; Germani, R.; Emiliani, C.; Tancini, B. Curcumin Analogue C1 Promotes Hex and Gal Recruitment to the Plasma Membrane via mTORC1-Independent TFEB Activation. Int. J. Mol. Sci. 2019, 20, 1363. https://doi.org/10.3390/ijms20061363
Magini A, Polchi A, Di Meo D, Buratta S, Chiaradia E, Germani R, Emiliani C, Tancini B. Curcumin Analogue C1 Promotes Hex and Gal Recruitment to the Plasma Membrane via mTORC1-Independent TFEB Activation. International Journal of Molecular Sciences. 2019; 20(6):1363. https://doi.org/10.3390/ijms20061363
Chicago/Turabian StyleMagini, Alessandro, Alice Polchi, Danila Di Meo, Sandra Buratta, Elisabetta Chiaradia, Raimondo Germani, Carla Emiliani, and Brunella Tancini. 2019. "Curcumin Analogue C1 Promotes Hex and Gal Recruitment to the Plasma Membrane via mTORC1-Independent TFEB Activation" International Journal of Molecular Sciences 20, no. 6: 1363. https://doi.org/10.3390/ijms20061363