Summary of study ST001320

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 PR000897. The data can be accessed directly via it's Project DOI: 10.21228/M8R704 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.

Perform statistical analysis  |  Show all samples  |  Show named metabolites  |  Download named metabolite data  |  Download all metabolite data  |  Download mwTab file (text)   |  Download mwTab file(JSON)   |  Download data (Contains raw data)
Study IDST001320
Study TitleExamination of labeled glucose utilization in central carbon metabolism in HeLa cells post WNT stimulation
Study TypeIn vitro
Study SummaryHeLa cells were treated with media containing either vehicle or WNT in combination with UC13-glucose for 20 min or 60 min.
Institute
University of California, Los Angeles
DepartmentBiological Chemisty
LaboratoryChristofk Lab
Last NameChristofk
First NameHeather
Address615 Charles E Young Drive South Los Angeles, CA 90095
Emailhchristofk@mednet.ucla.edu
Phone(310) 794-4248
Submit Date2020-02-19
Raw Data AvailableYes
Raw Data File Type(s).raw
Analysis Type DetailLC-MS
Release Date2020-07-18
Release Version1
Heather Christofk Heather Christofk
https://dx.doi.org/10.21228/M8R704
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:


Project:

Project ID:PR000897
Project DOI:doi: 10.21228/M8R704
Project Title:GSK3 inhibits macropinocytosis and lysosomal activity through the Wnt destruction complex machinery
Project Summary:Canonical Wnt signaling is emerging as a major regulator of endocytosis. Here we report that mutation of Axin1, a tumor-suppressor part of the β-catenin destruction complex, results in the activation of macropinocytosis in Alexander hepatocellular carcinoma (HCC) cells. Axin1 binds Glycogen Synthase Kinase 3 (GSK3), and we found that inhibition of GSK3 by Lithium chloride (LiCl), CHIR99021 or dominant-negative GSK3 triggered macropinocytosis. GSK3 inhibition caused a rapid increase in endolysosomes that was independent of new protein synthesis. GSK3 inhibition or Axin1 mutation increased lysosomal activity, which could be followed with tracers for active cathepsin D, β-glucosidase, and ovalbumin degradation. Microinjection of LiCl into the blastula cavity of Xenopus embryos caused a striking increase in dextran macropinocytosis. The effects of GSK3 inhibition on macropinocytosis and protein degradation in endolysosomes were blocked by the macropinocytosis inhibitors EIPA or IPA-2, suggesting that the increased membrane trafficking drives lysosomal activity and nutrient acquisition.
Institute:University of California, Los Angeles
Department:Biological Chemisty
Laboratory:De Robertis Lab
Last Name:De Robertis
First Name:Edward
Address:5-612A MRL
Email:ederobertis@mednet.ucla.edu
Phone:(310) 206-1401

Subject:

Subject ID:SU001394
Subject Type:Cultured cells
Subject Species:Homo sapiens
Taxonomy ID:9606

Factors:

Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id local_sample_id Treatment Treatment_Time(min)
SA095490HeLa-veh-20min-01vehicle 20
SA095491HeLa-veh-20min-03vehicle 20
SA095492HeLa-veh-20min-02vehicle 20
SA095493HeLa-veh-60min-03vehicle 60
SA095494HeLa-veh-60min-01vehicle 60
SA095495HeLa-veh-60min-02vehicle 60
SA095496HeLa-wnt-20min-03wnt 20
SA095497HeLa-wnt-20min-01wnt 20
SA095498HeLa-wnt-20min-02wnt 20
SA095499HeLa-wnt-60min-03wnt 60
SA095500HeLa-wnt-60min-02wnt 60
SA095501HeLa-wnt-60min-01wnt 60
Showing results 1 to 12 of 12

Collection:

Collection ID:CO001389
Collection Summary:Extraction began by placing the 6-well dishes on ice and washing each well with 2mL of a 150 mM ammonium acetate solution (pH 7.4, 4°C). Post wash, metabolites were extracted from each well on dry ice with 500 μL of a (1:4) dH20, MeOH solution at -80°C. The samples were then incubated for 15 min at -80°C. Afterwards, the cells were scraped into solutions which were then transferred to eppitubes and spun down at 4°C for 10 min at 17,000g. The supernatants were transferred to new glass vials and dried with an EZ2-Elite lyophilizer (Genevac). These dried metabolites were then stored at -80°C until they could be run on the LC/MS.
Sample Type:Cultured cells
Storage Conditions:Described in summary

Treatment:

Treatment ID:TR001409
Treatment Summary:The cells were incubated with DMEM(no glucose) supplemented with 10% dialyzed FBS, 1% P/S, and 10 mM UC13-glucose with either vehicle (0.1% BSA in dH20) or wnt (100ng/mL) added. Cells were treated for either 20 min or 60 min with each treatment group having a vehicle matched time control.

Sample Preparation:

Sampleprep ID:SP001402
Sampleprep Summary:Extraction began by placing the 6-well dishes on ice and washing each well with 2mL of a 150 mM ammonium acetate solution (pH 7.4, 4°C). Post wash, metabolites were extracted from each well on dry ice with 500 μL of a (1:4) dH20, MeOH solution at -80°C. The samples were then incubated for 15 min at -80°C. Afterwards, the metabolite solutions were transferred to eppitubes and spun down at 4°C for 10 min at 17,000g. The supernatants were transferred to new vials and dried with an EZ2-Elite lyophilizer (Genevac). These dried metabolites were then stored at -80°C until they could be run on the LC/MS.
Extract Storage:-80℃

Combined analysis:

Analysis ID AN002196
Analysis type MS
Chromatography type HILIC
Chromatography system Thermo Vanquish
Column Phenomenex Luna NH2 (150 x 2.1mm, 3um)
MS Type ESI
MS instrument type Orbitrap
MS instrument name Thermo Q Exactive Orbitrap
Ion Mode UNSPECIFIED
Units MS1 AUC integration

Chromatography:

Chromatography ID:CH001610
Chromatography Summary:Samples were run on a Vanquish (Thermo Scientific) UHPLC system with mobile phase A (5mM NH4AcO, pH 9.9) and mobile phase B (ACN) at a flow rate of 200 µL/min on a Luna 3 um NH2 100A (150 × 2.0mm) at 40°C with a gradient going from 15% A to 95% A in 18 min followed by an 11 minute isocratic step.
Instrument Name:Thermo Vanquish
Column Name:Phenomenex Luna NH2 (150 x 2.1mm, 3um)
Column Temperature:40
Flow Gradient:15% A to 95% A in 18 min followed by an 11 minute isocratic step
Flow Rate:200 µL/min
Solvent A:5mM NH4 acetate pH 9.9
Solvent B:acetonitrile
Chromatography Type:HILIC

MS:

MS ID:MS002042
Analysis ID:AN002196
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Method runs in polarity switching mode. Features were assigned via MzMine 2 and an in house list of MS1 and RT times.
Ion Mode:UNSPECIFIED
  logo