Influence of glial biotin availability on coping up with ER stress

Published: 20 Jan 2020 | Version 3 | DOI: 10.17632/gsd5hxdphy.3
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Description of this data

We hypothesize that biotin favors BCAA utilization and its deficiency accumulates BCAA to prolong autophagy inhibition and ER stress by chronic activation of mTORC1.

Experiment data files

  • Data 1- Invivo
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    We hypothesize that biotin favors BCAA utilization and lipogenesis while its deficiency accumulates BCAA to prolong autophagy inhibition and ER stress by chronic activation of mTORC1. The parameters of interest were evaluated in young and aged wistar rats. This dataset shows immunoblots of insulin-mTORC1 signaling, biotinylated carboxylase levels, ER stress, lipogenesis and autophagy markers. Enzymatic activities of biotin dependent carboxylases, intracellular amino acid content and Immunofluorescence images of young and aged rat brain sections to reveal the biotin availability in glial positive cells at provided in this data set. Further enzymatic assay of lipid content in brain cerebrum lysates are also given in this data set

    • Assay of biotinylated carboxylase activity
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      • ACC amd MCC
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      • PC
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    • Blot images
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      Loading Order : Y A Y A Y A Y A Y A , ( Y represents Young and A represents Aged)

      • Autophagy
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      • Biotinylated carboxylases and GCN2
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      • ER stress
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      • GAPDH and ponceau S
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      • Insulin Signalling AKT-mTOR downstream
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    • HPLC analysis of intracellular amino acid content
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    • Immunofluorescence
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    • Immunoprecipitation blots
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      Loading Order : Y A Y A Y A Y A Y A , ( Y represents Young and A represents Aged) Load order Invitro :Solvent control,0.5 microgram Tunicamycin,1 microgram tunicamycin,1T+Biotin,SC+Biotin

    • TG,Chol,Insulin assay
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      • Cholesterol
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  • Data 2 - Invitro
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    The significance of biotin availability to promote branched chain amino acid catabolism, influence mTORC1 signaling and thereby promote autophagy and lipogenesis under ER stress is evaluated in primary glial cultures. The Immunoblots of mTORC1 signaling, autophagy ER stress, lipogenesis and biotinylated carboxylases are represented. Intracellular amino acid levels were determined through HPLC and represented. Further the adaptive role of lipogenesis under ER stress were also evaluated using enzymatic assay of lipid accumulation and immunblots for lipogenesis markers.

    • Blots
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      Load order Solvent control,0.5 microgram Tunicamycin,1 microgram tunicamycin,1T+Biotin,SC+Biotin

    • Facs
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      Glia Stained with glial mareker GFAP-Alexa fluor 488 and DRAQ5 for nucleus flow cytometry BD Canto TM used

    • Glial culture images and confirmation
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      Validation of primary culture of rat glial cells. Primary culture of rat glial cells were performed by collagenase digestion of rat cerebrum and cultured in 25 mM glucose with 10% NCS. The validation was done by phase contrast imaging,staining with GFAP antibody and imaging in green channel and imaging in red channel confirming specific signals. Adipose derived stromal vascular cells and L6 myoblasts were used as negative controls. Phase contrast image of primary glial cells in culture, confluent glial cells, Hematoxylin stained glial cells, Adipose derived stromal vascular cells, L6 myoblast cells. GFAP positive staining in green channel was evaluated in F) p-formaldehyde fixed and methanol fixed glial cells which was absent in methanol fixed glial cells without GFAP primary antibody addition. Absence of GFAP positive staining was confirmed in negative control cells L6 cells with and without GFAP antibody addition. showing the corresponding red channel images of the Green channel images , All imaging was done at 20X magnification.

    • HPLC-Amino acid analysis
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      Loading order : Solvent control,0.5 microgram Tunicamycin,1 microgram tunicamycin,1T+Biotin,SC+Biotin

      • S1
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        • Lysate
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        • Medium
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      • S2
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        • Lysate
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        • Medium
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      • S3
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        • Lysate
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        • Medium
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    • Triglyceride and cholesterol content
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Steps to reproduce

Western Blotting:
Protein concentrations of tissue were determined based on Lowry’s protocol (Lowry et al., 1951) and equal concentrations of proteins were resolved by Gradient gel (5-15%) SDS-PAGE. The proteins were transferred to nitrocellulose membrane and blocked with 5% skimmed milk powder and fatty acid free bovine serum albumin for total and phosphorylated proteins, respectively. Following this the membranes were probed with respective primary and secondary antibodies at appropriate dilutions and developed using SuperSignal West Femto Chemiluminescent Substrate. The signals were recorded by LI-COR Odyssey Fc imager and analyzed using Image studio software version 5.2. The signals were normalized against respective GAPDH signals and/or total proteins stained with ponceau S.

Latest version

  • Version 3

    2020-01-20

    Published: 2020-01-20

    DOI: 10.17632/gsd5hxdphy.3

    Cite this dataset

    Ganesan, Dhasarathan; Jayavelu, Tamilselvan (2020), “Influence of glial biotin availability on coping up with ER stress ”, Mendeley Data, v3 http://dx.doi.org/10.17632/gsd5hxdphy.3

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Institutions

Anna University Chennai Centre for Biotechnology

Categories

Glia, Biotin, Cholesterol, Triacylglycerol, Mammalian Target of Rapamycin, Aging, Autophagy, Cellular Signaling, Insulin, Endoplasmic Reticulum Stress, Glial Cells

Licence

CC BY 4.0 Learn more

The files associated with this dataset are licensed under a Creative Commons Attribution 4.0 International licence.

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You can share, copy and modify this dataset so long as you give appropriate credit, provide a link to the CC BY license, and indicate if changes were made, but you may not do so in a way that suggests the rights holder has endorsed you or your use of the dataset. Note that further permission may be required for any content within the dataset that is identified as belonging to a third party.

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