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.
| Study ID | ST001320 |
| Study Title | Examination of labeled glucose utilization in central carbon metabolism in HeLa cells post WNT stimulation |
| Study Type | In vitro |
| Study Summary | HeLa 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 |
| Department | Biological Chemisty |
| Laboratory | Christofk Lab |
| Last Name | Christofk |
| First Name | Heather |
| Address | 615 Charles E Young Drive South Los Angeles, CA 90095 |
| hchristofk@mednet.ucla.edu | |
| Phone | (310) 794-4248 |
| Submit Date | 2020-02-19 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | raw(Thermo) |
| Analysis Type Detail | LC-MS |
| Release Date | 2020-07-18 |
| Release Version | 1 |
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) |
|---|---|---|---|
| SA095490 | HeLa-veh-20min-01 | vehicle | 20 |
| SA095491 | HeLa-veh-20min-03 | vehicle | 20 |
| SA095492 | HeLa-veh-20min-02 | vehicle | 20 |
| SA095493 | HeLa-veh-60min-03 | vehicle | 60 |
| SA095494 | HeLa-veh-60min-01 | vehicle | 60 |
| SA095495 | HeLa-veh-60min-02 | vehicle | 60 |
| SA095496 | HeLa-wnt-20min-03 | wnt | 20 |
| SA095497 | HeLa-wnt-20min-01 | wnt | 20 |
| SA095498 | HeLa-wnt-20min-02 | wnt | 20 |
| SA095499 | HeLa-wnt-60min-03 | wnt | 60 |
| SA095500 | HeLa-wnt-60min-02 | wnt | 60 |
| SA095501 | HeLa-wnt-60min-01 | wnt | 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: | 100% water; 5 mM ammonium acetate, pH 9.9 |
| Solvent B: | 100% 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 |
