{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST001320","ANALYSIS_ID":"AN002196","VERSION":"1","CREATED_ON":"February 21, 2020, 10:19 am"},

"PROJECT":{"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"},

"STUDY":{"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","EMAIL":"hchristofk@mednet.ucla.edu","PHONE":"(310) 794-4248"},

"SUBJECT":{"SUBJECT_TYPE":"Cultured cells","SUBJECT_SPECIES":"Homo sapiens","TAXONOMY_ID":"9606"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"HeLa-veh-20min-01",
"Factors":{"Treatment":"vehicle","Treatment_Time(min)":"20"},
"Additional sample data":{"RAW_FILE_NAME":"HeLa-veh-20min-01.RAW"}
},
{
"Subject ID":"-",
"Sample ID":"HeLa-veh-20min-02",
"Factors":{"Treatment":"vehicle","Treatment_Time(min)":"20"},
"Additional sample data":{"RAW_FILE_NAME":"HeLa-veh-20min-02.RAW"}
},
{
"Subject ID":"-",
"Sample ID":"HeLa-veh-20min-03",
"Factors":{"Treatment":"vehicle","Treatment_Time(min)":"20"},
"Additional sample data":{"RAW_FILE_NAME":"HeLa-veh-20min-03.RAW"}
},
{
"Subject ID":"-",
"Sample ID":"HeLa-veh-60min-01",
"Factors":{"Treatment":"vehicle","Treatment_Time(min)":"60"},
"Additional sample data":{"RAW_FILE_NAME":"HeLa-veh-60min-01.RAW"}
},
{
"Subject ID":"-",
"Sample ID":"HeLa-veh-60min-02",
"Factors":{"Treatment":"vehicle","Treatment_Time(min)":"60"},
"Additional sample data":{"RAW_FILE_NAME":"HeLa-veh-60min-02.RAW"}
},
{
"Subject ID":"-",
"Sample ID":"HeLa-veh-60min-03",
"Factors":{"Treatment":"vehicle","Treatment_Time(min)":"60"},
"Additional sample data":{"RAW_FILE_NAME":"HeLa-veh-60min-03.RAW"}
},
{
"Subject ID":"-",
"Sample ID":"HeLa-wnt-20min-01",
"Factors":{"Treatment":"wnt","Treatment_Time(min)":"20"},
"Additional sample data":{"RAW_FILE_NAME":"HeLa-wnt-20min-01.RAW"}
},
{
"Subject ID":"-",
"Sample ID":"HeLa-wnt-20min-02",
"Factors":{"Treatment":"wnt","Treatment_Time(min)":"20"},
"Additional sample data":{"RAW_FILE_NAME":"HeLa-wnt-20min-02.RAW"}
},
{
"Subject ID":"-",
"Sample ID":"HeLa-wnt-20min-03",
"Factors":{"Treatment":"wnt","Treatment_Time(min)":"20"},
"Additional sample data":{"RAW_FILE_NAME":"HeLa-wnt-20min-03.RAW"}
},
{
"Subject ID":"-",
"Sample ID":"HeLa-wnt-60min-01",
"Factors":{"Treatment":"wnt","Treatment_Time(min)":"60"},
"Additional sample data":{"RAW_FILE_NAME":"HeLa-wnt-60min-01.RAW"}
},
{
"Subject ID":"-",
"Sample ID":"HeLa-wnt-60min-02",
"Factors":{"Treatment":"wnt","Treatment_Time(min)":"60"},
"Additional sample data":{"RAW_FILE_NAME":"HeLa-wnt-60min-02.RAW"}
},
{
"Subject ID":"-",
"Sample ID":"HeLa-wnt-60min-03",
"Factors":{"Treatment":"wnt","Treatment_Time(min)":"60"},
"Additional sample data":{"RAW_FILE_NAME":"HeLa-wnt-60min-03.RAW"}
}
],
"COLLECTION":{"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_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."},

"SAMPLEPREP":{"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℃"},

"CHROMATOGRAPHY":{"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.","CHROMATOGRAPHY_TYPE":"HILIC","INSTRUMENT_NAME":"Thermo Vanquish","COLUMN_NAME":"Phenomenex Luna NH2 (150 x 2.1mm, 3um)","FLOW_GRADIENT":"15% A to 95% A in 18 min followed by an 11 minute isocratic step","FLOW_RATE":"200 µL/min","COLUMN_TEMPERATURE":"40","SOLVENT_A":"5mM NH4 acetate pH 9.9","SOLVENT_B":"acetonitrile"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS"},

"MS":{"INSTRUMENT_NAME":"Thermo Q Exactive Orbitrap","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"UNSPECIFIED","MS_COMMENTS":"Method runs in polarity switching mode. Features were assigned via MzMine 2 and an in house list of MS1 and RT times."},

"MS_METABOLITE_DATA":{
"Units":"MS1 AUC integration",

"Data":[{"Metabolite":"triflate","HeLa-veh-20min-01":"740000000.00","HeLa-veh-20min-02":"805000000.00","HeLa-veh-20min-03":"831000000.00","HeLa-veh-60min-01":"812000000.00","HeLa-veh-60min-02":"910000000.00","HeLa-veh-60min-03":"892000000.00","HeLa-wnt-20min-01":"770000000.00","HeLa-wnt-20min-02":"901000000.00","HeLa-wnt-20min-03":"762000000.00","HeLa-wnt-60min-01":"841000000.00","HeLa-wnt-60min-02":"914000000.00","HeLa-wnt-60min-03":"921000000.00"},{"Metabolite":"lactate","HeLa-veh-20min-01":"465471684.50","HeLa-veh-20min-02":"349942922.10","HeLa-veh-20min-03":"328189913.30","HeLa-veh-60min-01":"475612867.90","HeLa-veh-60min-02":"568416161.30","HeLa-veh-60min-03":"413571261.60","HeLa-wnt-20min-01":"641570260.80","HeLa-wnt-20min-02":"632609947.10","HeLa-wnt-20min-03":"519300318.50","HeLa-wnt-60min-01":"862498044.30","HeLa-wnt-60min-02":"748949225.60","HeLa-wnt-60min-03":"736625318.10"},{"Metabolite":"fumarate","HeLa-veh-20min-01":"24629725.88","HeLa-veh-20min-02":"20331506.88","HeLa-veh-20min-03":"24865391.20","HeLa-veh-60min-01":"20630026.81","HeLa-veh-60min-02":"22945642.06","HeLa-veh-60min-03":"20852399.05","HeLa-wnt-20min-01":"21487294.58","HeLa-wnt-20min-02":"27471617.33","HeLa-wnt-20min-03":"24124864.97","HeLa-wnt-60min-01":"22011334.55","HeLa-wnt-60min-02":"25497366.03","HeLa-wnt-60min-03":"24419215.41"},{"Metabolite":"succinate","HeLa-veh-20min-01":"130747846.60","HeLa-veh-20min-02":"97217647.54","HeLa-veh-20min-03":"92682634.18","HeLa-veh-60min-01":"101729533.70","HeLa-veh-60min-02":"173989117.10","HeLa-veh-60min-03":"151815362.20","HeLa-wnt-20min-01":"127570449.00","HeLa-wnt-20min-02":"125460533.40","HeLa-wnt-20min-03":"123345534.10","HeLa-wnt-60min-01":"114932577.10","HeLa-wnt-60min-02":"182406194.70","HeLa-wnt-60min-03":"177110035.50"},{"Metabolite":"taurine","HeLa-veh-20min-01":"1360896715.00","HeLa-veh-20min-02":"1502464711.00","HeLa-veh-20min-03":"1567806074.00","HeLa-veh-60min-01":"1513374766.00","HeLa-veh-60min-02":"1622229947.00","HeLa-veh-60min-03":"1600479234.00","HeLa-wnt-20min-01":"1082215455.00","HeLa-wnt-20min-02":"1567806074.00","HeLa-wnt-20min-03":"1437133262.00","HeLa-wnt-60min-01":"1317417595.00","HeLa-wnt-60min-02":"1567813510.00","HeLa-wnt-60min-03":"1709356722.00"},{"Metabolite":"L-aspartic acid","HeLa-veh-20min-01":"990743687.50","HeLa-veh-20min-02":"1078233244.00","HeLa-veh-20min-03":"1119481492.00","HeLa-veh-60min-01":"675423925.40","HeLa-veh-60min-02":"700715877.20","HeLa-veh-60min-03":"688486272.90","HeLa-wnt-20min-01":"856165464.50","HeLa-wnt-20min-02":"1099379779.00","HeLa-wnt-20min-03":"1020833631.00","HeLa-wnt-60min-01":"538504030.60","HeLa-wnt-60min-02":"651481918.50","HeLa-wnt-60min-03":"672474931.20"},{"Metabolite":"malate","HeLa-veh-20min-01":"383206489.90","HeLa-veh-20min-02":"383263569.20","HeLa-veh-20min-03":"404208962.10","HeLa-veh-60min-01":"314789741.80","HeLa-veh-60min-02":"368699431.10","HeLa-veh-60min-03":"330111181.30","HeLa-wnt-20min-01":"351751952.90","HeLa-wnt-20min-02":"467779006.30","HeLa-wnt-20min-03":"424853546.60","HeLa-wnt-60min-01":"349746225.80","HeLa-wnt-60min-02":"390266988.80","HeLa-wnt-60min-03":"420396338.30"},{"Metabolite":"phosphorylethanolamine","HeLa-veh-20min-01":"254062.34","HeLa-veh-20min-02":"0.00","HeLa-veh-20min-03":"202187.80","HeLa-veh-60min-01":"337189.74","HeLa-veh-60min-02":"434398.32","HeLa-veh-60min-03":"0.00","HeLa-wnt-20min-01":"0.00","HeLa-wnt-20min-02":"312894.15","HeLa-wnt-20min-03":"254053.16","HeLa-wnt-60min-01":"0.00","HeLa-wnt-60min-02":"316472.43","HeLa-wnt-60min-03":"430953.71"},{"Metabolite":"L-glutamine","HeLa-veh-20min-01":"7952017809.00","HeLa-veh-20min-02":"7705355281.00","HeLa-veh-20min-03":"7780151300.00","HeLa-veh-60min-01":"8230255514.00","HeLa-veh-60min-02":"9184237561.00","HeLa-veh-60min-03":"8177202660.00","HeLa-wnt-20min-01":"8294744527.00","HeLa-wnt-20min-02":"9817027957.00","HeLa-wnt-20min-03":"8659332452.00","HeLa-wnt-60min-01":"10438598525.00","HeLa-wnt-60min-02":"10449178560.00","HeLa-wnt-60min-03":"11146143176.00"},{"Metabolite":"L-glutamic acid","HeLa-veh-20min-01":"1990484737.00","HeLa-veh-20min-02":"2150743537.00","HeLa-veh-20min-03":"2236545608.00","HeLa-veh-60min-01":"2022523974.00","HeLa-veh-60min-02":"2150898811.00","HeLa-veh-60min-03":"2043757491.00","HeLa-wnt-20min-01":"1776237224.00","HeLa-wnt-20min-02":"2354101619.00","HeLa-wnt-20min-03":"2172210544.00","HeLa-wnt-60min-01":"1808384462.00","HeLa-wnt-60min-02":"2086850961.00","HeLa-wnt-60min-03":"2236363576.00"},{"Metabolite":"hydroxyglutaric acid","HeLa-veh-20min-01":"267009905.00","HeLa-veh-20min-02":"231827574.00","HeLa-veh-20min-03":"167711830.60","HeLa-veh-60min-01":"235036869.30","HeLa-veh-60min-02":"394176033.90","HeLa-veh-60min-03":"258491442.50","HeLa-wnt-20min-01":"892916969.30","HeLa-wnt-20min-02":"605649188.40","HeLa-wnt-20min-03":"442235634.00","HeLa-wnt-60min-01":"928292858.80","HeLa-wnt-60min-02":"618532992.10","HeLa-wnt-60min-03":"629200776.40"},{"Metabolite":"L-methionine","HeLa-veh-20min-01":"539642664.40","HeLa-veh-20min-02":"494753120.50","HeLa-veh-20min-03":"493628057.30","HeLa-veh-60min-01":"544080147.20","HeLa-veh-60min-02":"654060157.50","HeLa-veh-60min-03":"559824755.80","HeLa-wnt-20min-01":"555224070.40","HeLa-wnt-20min-02":"674185755.70","HeLa-wnt-20min-03":"572062357.20","HeLa-wnt-60min-01":"758389743.30","HeLa-wnt-60min-02":"747057509.50","HeLa-wnt-60min-03":"800916174.30"},{"Metabolite":"L-histidine","HeLa-veh-20min-01":"472586051.40","HeLa-veh-20min-02":"447668013.10","HeLa-veh-20min-03":"456314802.50","HeLa-veh-60min-01":"483407142.20","HeLa-veh-60min-02":"583187330.30","HeLa-veh-60min-03":"527901095.50","HeLa-wnt-20min-01":"495315383.70","HeLa-wnt-20min-02":"589652615.90","HeLa-wnt-20min-03":"512853841.30","HeLa-wnt-60min-01":"664399528.50","HeLa-wnt-60min-02":"661289325.10","HeLa-wnt-60min-03":"690440729.10"},{"Metabolite":"uric 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}

}