Quantification of Lipid Abundance and Evaluation of Lipid Distribution in &lt;em&gt;Caenorhabditis elegans&lt;/em&gt; by Nile Red and Oil Red O Staining

Escorcia, Wilber; Ruter, Dana L.; Nhan, James D.; Curran, Sean P.

doi:10.3791/57352

Cited by 85 publications

(83 citation statements)

References 13 publications

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“…Animals were illuminated with 10 minutes of continuous light exposure (XCite LED, 0.39 mW/mm 2 ) as described for O 2 consumption experiments (see C. elegans O 2 consumption section). Oil Red O (StatLab, McKinney, TX) staining was performed as described in previous protocols 44,45 . Briefly, Oil Red O product was diluted 3:2 in isopropanol, equilibrated for 2 days, and then filtered (0.4 μm).…”

Section: Methodsmentioning

confidence: 99%

“…Oil Red O (StatLab, McKinney, TX) staining was performed as described in previous protocols. 44,45 Briefly, Oil Red O product was diluted 3:2 in isopropanol, equilibrated for 2 days, and then filtered (0.4 μm). Immediately after light treatment, animals were dehydrated in 40% isopropanol/PBST (137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , Tween 20 0.1%) for 3 minutes.…”

Section: Whole Animal Fat Measurementmentioning

confidence: 99%

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Neuronal AMPK coordinates mitochondrial energy sensing and hypoxia resistance inC. elegans

et al. 2020

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Mitochondria are organelles that provide energy to cells. Matching energy supply with energy demand is coordinated through various processes and is critical for cellular adaptation and survival under changing conditions. Therefore, an organism's health depends not only on mitochondrial energy production, but on the ability to sense metabolic status and the ability of mitochondria to signal appropriately to use that energy. 1,2 The mechanisms through which cellular energy-related signaling can occur likely depend on the electrochemical gradient across the mitochondrial inner membrane (IM). Mitochondrial respiration results in a proton gradient across the IM known as the protonmotive force (PMF). Ultimately, respiratory complexes of the electron transport chain (ETC) convert chemical energy into electrical potential energy by pumping protons across the IM, creating this gradient. The PMF can then be consumed at ATP synthase,

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Section: Methodsmentioning

confidence: 99%

Section: Whole Animal Fat Measurementmentioning

confidence: 99%

Neuronal AMPK coordinates mitochondrial energy sensing and hypoxia resistance inC. elegans

et al. 2020

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“…VSMCs were cultured in 6-well plates and incubated with oxLDL for 72 hours. Afterward, the VSMCs were washed by PBS for twice followed by 15-minute 4% paraformaldehyde/PBS xation and then were stained by 100 ng/mL Nile Red for intracellular lipids detection [20]. All cell samples were observed and photographed microscopically (ZEISS LSM 800, Zeiss Microsystems).…”

Section: Assessment Of Intracellular Lipidsmentioning

confidence: 99%

Toll-Like Receptor 4 Mediated Oxidized Low-Density Lipoprotein-Induced Foam Cell Formation in Vascular Smooth Muscle Cells

Chen

Xue

Cao

et al. 2020

Preprint

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Background: Oxidized low-density lipoprotein (oxLDL) induced a foam-cell like phenotype of the vascular smooth muscle cells (VSMCs), leading to the inflammatory responses incorporating Toll-like receptors (Tlrs)-mediated cellular alterations. We previously found that Tlr4 participated in inflammation response in VSMCs under oxLDL stimulation. However, the role of Tlr4 in foam-cell formation and underlying molecular pathways has not been comprehensively elucidated. This study aimed to investigate the role of Tlr4-mediated mechanisms in oxLDL induced foam-cell formation within VSMCs. Methods: After incubated with different dose of oxLDL, the lipid, reactive oxygen species (ROS) accumulation and foam-cell phenotype of the VSMCs were detected. The alteration of Tlr family, ROS and lipid accumulation regulators including the Src kinase, Nox2, Nox4, Mnsod and sirtuins were measured. Then the Tlr4 was knock down and underlying cellular change and altered molecules were detected. Results: We showed that oxLDL induced foam-cell like phenotype in VSMCs and led to lipid and ROS accumulation in a dose-dependent manner. OxLDL induced the strongest upregulation of Tlr4 in the Tlrs family and initiated change of Src activation, Nox2, Mnsod, sirt1 and sirt3 expression. The effect of oxLDL was abolished by Tlr4 knockdown. Furthermore, knocking down of Tlr4 reduced Src activation and led to restored Sirt1/Sirt3 expression. Moreover, inhibiting or knocking down the Src kinase diminished lipid accumulation in VSMCs under oxLDL treatment. And overexpression of Sirt1/3 relieved the oxLDL induced ROS accumulation and foam-cell phenotype in VSMCs.Conclusions: These results demonstrated that Tlr4 is a critical regulator in oxLDL induced foam cell formation of VSMCs via mediating Src kinase as well as Sirt1 and Sirt3. Beyond the role of Tlr4 in inflammation response of VSMCs, we provide an integrated mechanism about TLR4 in VSMCs phenotype transition under oxLDL stimulation.

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“…Synchronous L4 animals were stained as in 73 . Briefly, animals were washed from plate, fixed in 40% isopropanol for 3 min, stained in 0.03 mg/ml Nile Red in 40% isopropanol for 2 h, and de-stained in PBS with 0.01% Triton for 30 min.…”

Section: Methodsmentioning

confidence: 99%

Roles for the RNA polymerase III regulator MAFR-1 in regulating sperm quality in Caenorhabditis elegans

Hammerquist¹,

Curran²

2020

Sci Rep

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The negative regulator of RNA polymerase (pol) III mafr-1 has been shown to affect RNA pol III transcript abundance, lipid biosynthesis and storage, progeny output, and lifespan. We deleted mafr-1 from the Caenorhabditis elegans genome and found that animals lacking mafr-1 replicated many phenotypes from previous RNAi-based studies and discovered a new sperm-specific role. Utilizing a yeast two-hybrid assay, we discovered several novel interactors of MAFR-1 that are expressed in a sperm- and germline-enriched manner. In support of a role for MAFR-1 in the male germline, we found mafr-1 null males have smaller spermatids that are less capable in competition for fertilization; a phenotype that was dependent on RNA pol III activity. Restoration of MAFR-1 expression specifically in the germline rescued the spermatid-related phenotypes, suggesting a cell autonomous role for MAFR-1 in nematode male fertility. Based on the high degree of conservation of Maf1 activity across species, our study may inform similar roles for Maf1 and RNA pol III in mammalian male fertility.

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Quantification of Lipid Abundance and Evaluation of Lipid Distribution in <em>Caenorhabditis elegans</em> by Nile Red and Oil Red O Staining

Cited by 85 publications

References 13 publications

Neuronal AMPK coordinates mitochondrial energy sensing and hypoxia resistance inC. elegans

Neuronal AMPK coordinates mitochondrial energy sensing and hypoxia resistance inC. elegans

Toll-Like Receptor 4 Mediated Oxidized Low-Density Lipoprotein-Induced Foam Cell Formation in Vascular Smooth Muscle Cells

Roles for the RNA polymerase III regulator MAFR-1 in regulating sperm quality in Caenorhabditis elegans

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