Summary of Study ST001708

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001093. The data can be accessed directly via it's Project DOI: 10.21228/M8DT31 This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

Study ID	ST001708
Study Title	Oxylipin biosynthesis reinforces cellular senescence through a RAS/p53 feedback loop and allows detection of senolysis
Study Summary	Cellular senescence is a stress or damage response that causes a permanent proliferative arrest and secretion of numerous factors with potent biological activities. This senescence-associated secretory phenotype (SASP) has been characterized largely for secreted proteins that participate in embryogenesis, wound healing, inflammation and many age-related pathologies. By contrast, lipid components of the SASP are understudied. We show that senescent cells activate the biosynthesis of several oxylipins that promote segments of the SASP and reinforce the proliferative arrest. Notably, senescent cells synthesize and accumulate an unstudied intracellular prostaglandin, 1a,1b-dihomo-15-deoxy-delta-12,14-prostaglandin J2. Released 15-deoxy-delta-12,14-prostaglandin J2 is a biomarker of senolysis in culture and in vivo. This and other prostaglandin D2-related lipids promote the senescence arrest and SASP by activating RAS signaling. These data identify an important aspect of cellular senescence and a method to detect senolysis
Institute	Buck Institute for Research on Aging
Last Name	Sharma
First Name	Rishi
Address	8001 Redwood Blvd, Novato, CA, 94945, USA
Email	sharmarishi2004@yahoo.co.in
Phone	5084392367
Submit Date	2021-02-20
Raw Data Available	Yes
Raw Data File Type(s)	d
Analysis Type Detail	LC-MS
Release Date	2021-08-10
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR001093
Project DOI:	doi: 10.21228/M8DT31
Project Title:	Oxylipin biosynthesis reinforces cellular senescence through a RAS/p53 feedback loop and allows detection of senolysis
Project Summary:	Cellular senescence is a stress or damage response that causes a permanent proliferative arrest and secretion of numerous factors with potent biological activities. This senescence-associated secretory phenotype (SASP) has been characterized largely for secreted proteins that participate in embryogenesis, wound healing, inflammation and many age-related pathologies. By contrast, lipid components of the SASP are understudied. We show that senescent cells activate the biosynthesis of several oxylipins that promote segments of the SASP and reinforce the proliferative arrest. Notably, senescent cells synthesize and accumulate an unstudied intracellular prostaglandin, 1a,1b-dihomo-15-deoxy-delta-12,14-prostaglandin J2. Released 15-deoxy-delta-12,14-prostaglandin J2 is a biomarker of senolysis in culture and in vivo. This and other prostaglandin D2-related lipids promote the senescence arrest and SASP by activating RAS signaling. These data identify an important aspect of cellular senescence and a method to detect senolysis.
Institute:	Buck Institute for Research on Aging
Last Name:	Sharma
First Name:	Rishi
Address:	8001 Redwood Blvd, Novato, CA, 94945, USA
Email:	sharmarishi2004@yahoo.co.in
Phone:	5084392367

Subject:

Subject ID:	SU001785
Subject Type:	Cultured cells
Subject Species:	Mus musculus
Taxonomy ID:	10090

Factors:

Subject type: Cultured cells; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id	local_sample_id	Factor
SA159037	IR_DOXOABT_1	DOXOABT
SA159038	A-121	Mouse plasma
SA159039	Ctl_61120	SEN Cells_Ctls
SA159040	IR_15_60120	SENCells_IR

Showing results 1 to 4 of 4

Showing results 1 to 4 of 4

Collection:

Collection ID:	CO001778
Collection Summary:	Cell culture: Human fetal lung fibroblasts (IMR-90) and HEPG2 cells were cultured in Dulbecco’s modified eagle medium (DMEM - Gibco) supplemented with 10% FBS and penicillin/streptomycin (Gibco). HUVEC cells were obtained from the ATCC and cultured using the ATCC protocol and culture media. Quiescence was induced by replacing culture media with media containing 0.2% FBS for 72 h before analysis. All cells were cultured at 3% O2, and used prior to 40 population doublings, other than replicative senescent cells, which were cultured until replicative exhaustion. All cells were mycoplasma free. Mouse Plasma: Animal experiments were conducted using a protocol approved by the Institutional Animal Care and Use Committee of the Buck Institute. For DOXO treatments, 10-16 wk old p16-3MR mice received one intraperitoneal (i.p.) injection of 10 mg/kg of doxorubicin hydrochloride in PBS, and treated 5 d later with GCV or vehicle. GCV was administered via daily i.p. injections for 5 consecutive days at 25 mg/kg in PBS. Control mice were injected with an equal volume of PBS. Mice were euthanized and tissues collected 10 d after DOXO challenge. For aging studies, C57BL/6 mice were aged for 6 or 24 mo, at which point mice were euthanized and tissues collected for analysis. For the biomarker studies, mice with challenged with DOXO as above, and given either vehicle or ABT-263 6 weeks after DOXO challenge. Whole blood was collected by cardiac puncture into EDTA-tubes (lavender caps – BD Biosciences) and spun at 2000g for 5 minutes. For urine collection, mice were placed in plastic containers prior to euthanasia.
Sample Type:	Blood (plasma)

Treatment:

Treatment ID:	TR001798
Treatment Summary:	Cell culture: Human fetal lung fibroblasts (IMR-90) and HEPG2 cells were cultured in Dulbecco’s modified eagle medium (DMEM - Gibco) supplemented with 10% FBS and penicillin/streptomycin (Gibco). HUVEC cells were obtained from the ATCC and cultured using the ATCC protocol and culture media. Quiescence was induced by replacing culture media with media containing 0.2% FBS for 72 h before analysis. All cells were cultured at 3% O2, and used prior to 40 population doublings, other than replicative senescent cells, which were cultured until replicative exhaustion. All cells were mycoplasma free. Mouse Plasma: Animal experiments were conducted using a protocol approved by the Institutional Animal Care and Use Committee of the Buck Institute. For DOXO treatments, 10-16 wk old p16-3MR mice received one intraperitoneal (i.p.) injection of 10 mg/kg of doxorubicin hydrochloride in PBS, and treated 5 d later with GCV or vehicle. GCV was administered via daily i.p. injections for 5 consecutive days at 25 mg/kg in PBS. Control mice were injected with an equal volume of PBS. Mice were euthanized and tissues collected 10 d after DOXO challenge. For aging studies, C57BL/6 mice were aged for 6 or 24 mo, at which point mice were euthanized and tissues collected for analysis. For the biomarker studies, mice with challenged with DOXO as above, and given either vehicle or ABT-263 6 weeks after DOXO challenge. Whole blood was collected by cardiac puncture into EDTA-tubes (lavender caps – BD Biosciences) and spun at 2000g for 5 minutes. For urine collection, mice were placed in plastic containers prior to euthanasia.

Treatment ID:

TR001798

Treatment Summary:

Cell culture: Human fetal lung fibroblasts (IMR-90) and HEPG2 cells were cultured in Dulbecco’s modified eagle medium (DMEM - Gibco) supplemented with 10% FBS and penicillin/streptomycin (Gibco). HUVEC cells were obtained from the ATCC and cultured using the ATCC protocol and culture media. Quiescence was induced by replacing culture media with media containing 0.2% FBS for 72 h before analysis. All cells were cultured at 3% O2, and used prior to 40 population doublings, other than replicative senescent cells, which were cultured until replicative exhaustion. All cells were mycoplasma free. Mouse Plasma: Animal experiments were conducted using a protocol approved by the Institutional Animal Care and Use Committee of the Buck Institute. For DOXO treatments, 10-16 wk old p16-3MR mice received one intraperitoneal (i.p.) injection of 10 mg/kg of doxorubicin hydrochloride in PBS, and treated 5 d later with GCV or vehicle. GCV was administered via daily i.p. injections for 5 consecutive days at 25 mg/kg in PBS. Control mice were injected with an equal volume of PBS. Mice were euthanized and tissues collected 10 d after DOXO challenge. For aging studies, C57BL/6 mice were aged for 6 or 24 mo, at which point mice were euthanized and tissues collected for analysis. For the biomarker studies, mice with challenged with DOXO as above, and given either vehicle or ABT-263 6 weeks after DOXO challenge. Whole blood was collected by cardiac puncture into EDTA-tubes (lavender caps – BD Biosciences) and spun at 2000g for 5 minutes. For urine collection, mice were placed in plastic containers prior to euthanasia.

Sample Preparation:

Sampleprep ID:	SP001791
Sampleprep Summary:	Protocol for Lipid Extraction (10.24.2019) Lipid Biomarker for Senolysis Study 1. 80% MeOH was added to each sample 500µL to plasma samples 100µL to urine sample 2. Transfer the contents to 9 mL glass tube and add 1.0 mL of CHCl3 (containing 200 ng/mL FA 17:0 Saturated). Vortex mix for 5 minutes. 3. Place the glass tube into a 15mL polypropylene culture tube with Kimi wipes inserted at bottom as a cushion. Wrap the culture tube with paraffin and place in centrifuge. This will prevent glass tube being shattered in the centrifuge. 4. Centrifuge at 4°C for 10 minutes 3000rpm. 5. After spinning there will be two distinct layers, the top layer is the aqueous layer containing the aqueous metabolites (80% MeOH) and bottom layer which contains lipids. 6. Transfer the lower layer into 1.5ml vials. 7. Transfer from the 1.5mL tube the exact amount of each sample into the MS/HPLC glass vial and dry down using nitrogen gas. 8. Reconstitute the lipids with 80µL chloroform. 9. Transfer upper layer (metabolites) into labeled vials and store at -80°C, if possible. Samples stored in -80°C. Sample volume submitted -Plasma samples=50µL Urine samples= 10µL Sample volume in each vial- Plasma samples µL and Urine samples µL -submitted to mass spec All samples were split in half ½ given to CW for further testing other 1/2 in -80°C

Sampleprep ID:

SP001791

Sampleprep Summary:

Protocol for Lipid Extraction (10.24.2019) Lipid Biomarker for Senolysis Study 1. 80% MeOH was added to each sample 500µL to plasma samples 100µL to urine sample 2. Transfer the contents to 9 mL glass tube and add 1.0 mL of CHCl3 (containing 200 ng/mL FA 17:0 Saturated). Vortex mix for 5 minutes. 3. Place the glass tube into a 15mL polypropylene culture tube with Kimi wipes inserted at bottom as a cushion. Wrap the culture tube with paraffin and place in centrifuge. This will prevent glass tube being shattered in the centrifuge. 4. Centrifuge at 4°C for 10 minutes 3000rpm. 5. After spinning there will be two distinct layers, the top layer is the aqueous layer containing the aqueous metabolites (80% MeOH) and bottom layer which contains lipids. 6. Transfer the lower layer into 1.5ml vials. 7. Transfer from the 1.5mL tube the exact amount of each sample into the MS/HPLC glass vial and dry down using nitrogen gas. 8. Reconstitute the lipids with 80µL chloroform. 9. Transfer upper layer (metabolites) into labeled vials and store at -80°C, if possible. Samples stored in -80°C. Sample volume submitted -Plasma samples=50µL Urine samples= 10µL Sample volume in each vial- Plasma samples µL and Urine samples µL -submitted to mass spec All samples were split in half ½ given to CW for further testing other 1/2 in -80°C

Combined analysis:

Analysis ID	AN002782
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Agilent 6520
Column	Phenomenex Kinetex C18 (150 x 2.1mm,2.6um)
MS Type	ESI
MS instrument type	QToF
MS instrument name	Agilent 6520 QTOF
Ion Mode	NEGATIVE
Units	Peak Area

Chromatography:

Chromatography ID:	CH002058
Instrument Name:	Agilent 6520
Column Name:	Phenomenex Kinetex C18 (150 x 2.1mm,2.6um)
Chromatography Type:	Reversed phase

MS:

MS ID:	MS002578
Analysis ID:	AN002782
Instrument Name:	Agilent 6520 QTOF
Instrument Type:	QToF
MS Type:	ESI
MS Comments:	Full scan mode was used. Data processed using profinder software
Ion Mode:	NEGATIVE