Functional Mitochondria Are Important for the Effect of Resveratrol
"> Figure 1
<p>Effect of Resveratrol on cell number, proliferation and cell diameter. (<b>A</b>) normalized cell counts of HeLa wild type (WT) and HeLa Rho 0 cells treated with Resv (10 and 50 µM for 24 and 48 h). HeLa WT 10, 50 µM Resv vs. HeLa Rho 0 10, 50 µM Resv, <span class="html-italic">p</span> < 0.001 (***) at 24 h Cell counts in HeLa WT 10 µM Resv vs. HeLa Rho 0 10 µM Resv, <span class="html-italic">p</span> < 0.01 (**) and HeLa WT 50 µM Resv vs. HeLa Rho 0 50 µM Resv, <span class="html-italic">p</span> < 0.05 (*) at 48 h; (<b>B</b>) impedance curves of HeLa WT and Rho 0 cells treated with Resv in long-term exposure (110 h); and (<b>C</b>) cell diameter in HeLa WT and HeLa Rho 0 cells treated for 24 h with 5–40 µM Resv. HeLa WT compared with Rho 0 when treated with 5 to 40 µM Resv, <span class="html-italic">p</span> < 0.01(**). All values are a pool of three independent experiments with a determination of four replicates in (<b>A</b>), (<b>C</b>) and two replicates in (<b>B</b>). ANOVA/Bonferroni used for statistical analysis.</p> "> Figure 1 Cont.
<p>Effect of Resveratrol on cell number, proliferation and cell diameter. (<b>A</b>) normalized cell counts of HeLa wild type (WT) and HeLa Rho 0 cells treated with Resv (10 and 50 µM for 24 and 48 h). HeLa WT 10, 50 µM Resv vs. HeLa Rho 0 10, 50 µM Resv, <span class="html-italic">p</span> < 0.001 (***) at 24 h Cell counts in HeLa WT 10 µM Resv vs. HeLa Rho 0 10 µM Resv, <span class="html-italic">p</span> < 0.01 (**) and HeLa WT 50 µM Resv vs. HeLa Rho 0 50 µM Resv, <span class="html-italic">p</span> < 0.05 (*) at 48 h; (<b>B</b>) impedance curves of HeLa WT and Rho 0 cells treated with Resv in long-term exposure (110 h); and (<b>C</b>) cell diameter in HeLa WT and HeLa Rho 0 cells treated for 24 h with 5–40 µM Resv. HeLa WT compared with Rho 0 when treated with 5 to 40 µM Resv, <span class="html-italic">p</span> < 0.01(**). All values are a pool of three independent experiments with a determination of four replicates in (<b>A</b>), (<b>C</b>) and two replicates in (<b>B</b>). ANOVA/Bonferroni used for statistical analysis.</p> "> Figure 2
<p>Mitochondrial activity of HeLa WT and HeLa Rho 0 following 24 h exposure to resveratrol. (<b>A</b>) Oxygen consumption rates (OCR), HeLa WT trace; (<b>B</b>) OCR, HeLa Rho 0 trace; (<b>C</b>) average of basal respiration measurements, HeLa WT and Rho 0, were HeLa WT 20 and 30 µM Resv compared to HeLa WT Ctrl (*: <span class="html-italic">p</span> < 0.05); (<b>D</b>) relative OCR related to ATP production of HeLa WT and Rho 0 calculated data after addition of oligomycin, were HeLa WT ctrl compared to HeLa Rho 0 Ctrl (***: <span class="html-italic">p</span> < 0.001); (<b>E</b>) relative rate of non-mitochondrial respiration of HeLa WT and Rho 0, calculated data after addition of rotenone/antimycin A, were HeLa WT ctrl compared to HeLa Rho 0 Ctrl (***: <span class="html-italic">p</span> < 0.001); (<b>F</b>) relative OCR related to proton leak of HeLa WT and Rho 0 calculated after addition of oligomycin minus non mitochondrial respiration. Data are presented as mean of three experiments ± SEM. ANOVA/Bonferroni used for statistical analysis.</p> "> Figure 3
<p>Mitochondrial membrane potential and cellular reactive oxygen species in HeLa WT and Rho 0 cells. Cells were treated with or without 20 µM Resv for 24 h, and mitochondria were labeled using (<b>A</b>) Tetramethylrhodamine, methyl ester, perchlorate (TMRM), as an estimate of mitochondrial membrane potential; (<b>B</b>) H<sub>2</sub>DCFDA to visualize reactive oxygen species. The estimates are shown as relative to a simultanous estimate of mitochondrial mass by Mitotracker green (MTG). Data are presented as mean ± SEM (<span class="html-italic">n</span> = 3) analyzed using <span class="html-italic">t</span>-test.</p> "> Figure 4
<p>Glycolytic activity of HeLa WT and HeLa Rho 0 following 24 h exposure to resveratrol. (<b>A</b>) extracellular acidification rate (ECAR), HeLa WT trace; (<b>B</b>) ECAR, HeLa Rho 0 trace; (<b>C</b>) average of basal ECAR, HeLa WT and Rho 0; (<b>D</b>) relative ECAR of HeLa WT and Rho 0 determined after oligomycin addition. Data are presented as mean of three experiments ± SEM.</p> "> Figure 5
<p>Effect of Resveratrol on expression levels of mRNA transcripts related to mitochondrial function. Relative gene expression of key genes related to mitochondrial function measured by Q-RT-PCR (quantitative reverse transcriptase polymerase chain reaction) in HeLa WT and HeLa Rho 0 cells treated with 10 and 20 µM Resv for 24 h. Expression of (<b>A</b>) NAD (nicotineamide dinucleotide)-dependent deacetylase sirtuin-1 (SIRT1); (<b>B</b>) mitochondrial transcription factor A (TFAM), HeLa WT compared to HeLa Rho 0 (***: <span class="html-italic">p</span> < 0.001) untreated; (<b>C</b>) nuclear respiratory factor 1 (NRF-1 also known as NFE2L1), HeLa WT compared to HeLa Rho 0 (***: <span class="html-italic">p</span> < 0.001) untreated and HeLa Rho 0 untreated compared to HeLa Rho 0 10 µM Resv (*: <span class="html-italic">p</span> < 0.05); (<b>D</b>) cytochrome C oxidase 5b (COX5b); (<b>E</b>) estrogen-related receptor alpha (ERR-α), HeLa WT compared to HeLa Rho 0 (***: <span class="html-italic">p</span> < 0.001) untreated and HeLa Rho 0 untreated compared to HeLa Rho 0 10 µM Resv (*: <span class="html-italic">p</span> < 0.05), compared to 20 µM (***: <span class="html-italic">p</span> < 0.001); (<b>F</b>) cytochrome complex (Cyt C), HeLa WT compared to HeLa Rho 0 (***: <span class="html-italic">p</span> < 0.001) untreated, (<b>G</b>) proliferator-activated receptor coactivator-1α (PGC-1α). All data are presented as relative to levels of RPL23a and are shown as mean ± SEM (<span class="html-italic">n</span> = 6). Statistical analysis performed using a <span class="html-italic">t</span>-test.</p> "> Figure 5 Cont.
<p>Effect of Resveratrol on expression levels of mRNA transcripts related to mitochondrial function. Relative gene expression of key genes related to mitochondrial function measured by Q-RT-PCR (quantitative reverse transcriptase polymerase chain reaction) in HeLa WT and HeLa Rho 0 cells treated with 10 and 20 µM Resv for 24 h. Expression of (<b>A</b>) NAD (nicotineamide dinucleotide)-dependent deacetylase sirtuin-1 (SIRT1); (<b>B</b>) mitochondrial transcription factor A (TFAM), HeLa WT compared to HeLa Rho 0 (***: <span class="html-italic">p</span> < 0.001) untreated; (<b>C</b>) nuclear respiratory factor 1 (NRF-1 also known as NFE2L1), HeLa WT compared to HeLa Rho 0 (***: <span class="html-italic">p</span> < 0.001) untreated and HeLa Rho 0 untreated compared to HeLa Rho 0 10 µM Resv (*: <span class="html-italic">p</span> < 0.05); (<b>D</b>) cytochrome C oxidase 5b (COX5b); (<b>E</b>) estrogen-related receptor alpha (ERR-α), HeLa WT compared to HeLa Rho 0 (***: <span class="html-italic">p</span> < 0.001) untreated and HeLa Rho 0 untreated compared to HeLa Rho 0 10 µM Resv (*: <span class="html-italic">p</span> < 0.05), compared to 20 µM (***: <span class="html-italic">p</span> < 0.001); (<b>F</b>) cytochrome complex (Cyt C), HeLa WT compared to HeLa Rho 0 (***: <span class="html-italic">p</span> < 0.001) untreated, (<b>G</b>) proliferator-activated receptor coactivator-1α (PGC-1α). All data are presented as relative to levels of RPL23a and are shown as mean ± SEM (<span class="html-italic">n</span> = 6). Statistical analysis performed using a <span class="html-italic">t</span>-test.</p> ">
Abstract
:1. Introduction
2. Results
2.1. Effect of Resveratrol on Cell Numbers, Proliferation and Diameter
2.2. Effect of Resveratrol Exposure for 24 h on Oxygen Consumption Rate in HeLa Cells
2.3. Resveratrol Effect on Mitochondrial Mass, Membrane Potential and Reactive Oxygen Species in HeLa WT and Rho 0 Cells
2.4. Effect of Resveratrol Exposure for 24 h on Extracellular Acidification Rate (ECAR)
2.5. Effect of Resveratrol on Mitochondria Related mRNA Transcript Levels
3. Discussion
4. Materials and Methods
4.1. Materials
4.2. Cell Culture
4.3. Determination of Cell Number and Size
4.4. Impedance Readings
4.5. Respirometry in Intact Cells
4.6. Detection of Mitochondrial Mass, Mitochondrial Membrane Potential and Oxidative Stress by Flow Cytometry
4.7. Quantitative Real Time RT-PCR Assay
4.8. Statistical Analysis
5. Conclusions
Supplementary Materials
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Widlund, A.L.; Baral, K.; Dalgaard, L.T.; Vang, O. Functional Mitochondria Are Important for the Effect of Resveratrol. Molecules 2017, 22, 847. https://doi.org/10.3390/molecules22050847
Widlund AL, Baral K, Dalgaard LT, Vang O. Functional Mitochondria Are Important for the Effect of Resveratrol. Molecules. 2017; 22(5):847. https://doi.org/10.3390/molecules22050847
Chicago/Turabian StyleWidlund, Anne L., Kaushal Baral, Louise T. Dalgaard, and Ole Vang. 2017. "Functional Mitochondria Are Important for the Effect of Resveratrol" Molecules 22, no. 5: 847. https://doi.org/10.3390/molecules22050847